A disintegrin and metalloproteinase with a thrombospondin type i motif, member 13 (adamts-13) mutants, compositions and therapeutic methods thereof

ABSTRACT

The present invention relates to ADAMTS-13 mutants and/or variant/s that display resistance to tPA cleavage and/or inactivation. The present disclosure further provides fibrinolytic compounds, compositions, combined compositions and kits comprising the ADAMTS-13 mutants disclosed herein, as well as uses thereof for treating coagulation related disorders.

FIELD OF THE INVENTION

The invention relates to coagulation modulators. More specifically, theinvention relates to disintegrin and metalloproteinase with athrombospondin type 1 motif, member 13 (ADAMTS-13) mutants and/orvariant, compositions and uses thereof for treating coagulation relateddisorders.

BACKGROUND ART

References considered to be relevant as background to the presentlydisclosed subject matter are listed below:

-   [1] Bao J, Xiao J, Mao Y, Zheng X L. Arterioscler Thromb Vasc Biol    2014, 34(2): 397-407, Carboxyl terminus of ADAMTS-13 directly    inhibits platelet aggregation and ultra large von Willebrand factor    string formation under flow in a free-thiol-dependent manner.-   [2] Galbusera M, Noris M, Remuzzi G. Semin Thromb Hemost 2006 32(2):    81-89, Thrombotic thrombocytopenic purpura—then and now.-   [3] Bongers T N, de Maat M P, van Goor M L, Bhagwanbali V, van Vliet    H H, Gómez Garcia E B, Dippel D W, Leebeek F W. Stroke. 2006    November; 37(11):2672-7; High von Willebrand factor levels increase    the risk of first ischemic stroke: influence of ADAMTS-13,    inflammation, and genetic variability.-   [4] Galbusera M, Noris M, Remuzzi G. Semin Thromb Hemost. 2006    March; 32(2):81-9. Review. Thrombotic thrombocytopenic purpura—then    and now.-   [5] Zhao B Q, Chauhan A K, Canault M, Patten I S, Yang J J, Dockal    M, Scheiflinger F, Wagner D D. Blood. 2009 Oct. 8; 114(15):3329-34.    von Willebrand factor-cleaving protease ADAMTS-13 reduces ischemic    brain injury in experimental stroke.-   [6] Zeng M, Chen Q, Liang W, He W, Zheng H, Huang C, Int J Chron    Obstruct Pulmon Dis. 2017 Dec. 5; 12:3495-3501. Predictive value of    ADAMTS-13 on concealed chronic renal failure in COPD patients.-   [7] Denorme F, Langhauser F, Desender L, Vandenbulcke A,    Rottensteiner H, Plaimauer B, François O, Andersson T, Deckmyn H,    Scheiflinger F, Kleinschnitz C, Vanhoorelbeke K, De Meyer S F.    Blood. 2016 May 12; 127(19):2337-45. doi:    10.1182/blood-2015-08-662650. Epub 2016 Feb. 29. ADAMTS-13-mediated    thrombolysis of t-P A-resistant occlusions in ischemic stroke in    mice.-   [8] José A. Diaz, Angela E. Hawley, Christine M. Alvarado,    Alexandra M. Berguer, Nichole K. Baker, Shirley K. Wrobleski,    Thomas W. Wakefield, Benedict R. Lucchesi, and Daniel D. Myers, Jr.    Thromb Haemost 2010; 104: 366-375, Thrombogenesis with continuous    blood flow in the inferior vena cava: A novel mouse model.-   [9] Denorme F, Langhauser F, Desender L, Vandenbulcke A,    Rottensteiner H, Plaimauer B, François O, Andersson T, Deckmyn H,    Scheiflinger F, Kleinschnitz C, Vanhoorelbeke K, De Meyer S F. Blood    2016 127:2337-2345, ADAMTS-13-mediated thrombolysis of t-P    A-resistant occlusions in ischemic stroke in mice.-   [10] Schuhmann M K, Gunreben I, Kleinschnitz C, Kraft P. Int J Mol    Sci 2016 17(3), Int J Mol Sci. 2016; 17(3):298. Immunohistochemical    Analysis of Cerebral Thrombi Retrieved by Mechanical Thrombectomy    from Patients with Acute Ischemic Stroke.-   [11] Miszta A, Pelkmans L, Lindhout T, Krishnamoorthy G, de Groot P    G, Hemker C H, Heemskerk J W, Kelchtermans H, de Laat B. J Biol Chem    2014, 289(52): 35979-35986, Thrombin-dependent Incorporation of von    Willebrand Factor into a Fibrin Network.-   [12] Bhatia R, Hill M D, Shobha N, Menon B, Bal S, Kochar P, Watson    T, Goyal M, Demchuk A M. Stroke 2010, 41(10): 2254-2258, Low rates    of acute recanalization with intravenous recombinant tissue    plasminogen activator in ischemic stroke: real-world experience and    a call for action.-   [13] Kleinschnitz C, De Meyer S F, Schwarz T, Austinat M,    Vanhoorelbeke K, Nieswandt B, Deckmyn H, Stoll G. Blood 2009    113(15): 3600-3603, Deficiency of von Willebrand factor protects    mice from ischemic stroke.-   [14] Kleinschnitz C¹, De Meyer S F, Schwarz T, Austinat M,    Vanhoorelbeke K, Nieswandt B, Deckmyn H, Stoll G. Blood. 2009 Apr.    9; 113(15):3600-3. Deficiency of von Willebrand factor protects mice    from ischemic stroke.-   [15] Fujioka M, Hayakawa K, Mishima K, Kunizawa A, Irie K, Higuchi    S, Nakano T, Muroi C, Fukushima H, Sugimoto M, Banno F, Kokame K,    Miyata T, Fujiwara M, Okuchi K, Nishio K. Blood 2010 115(8):    1650-1653, ADAMTS-13 gene deletion aggravates ischemic brain damage:    a possible neuroprotective role of ADAMTS-13 by ameliorating    postischemic hypoperfusion.-   [16] Sonneveld M A, de Maat M P, Portegies M L, Kavousi M, Hofman A,    Turecek P L, Rottensteiner H, Scheiflinger F, Koudstaal P J, Ikram M    A, Leebeek F W. Blood 2015 126(25): 2739-2746. Low ADAMTS-13    activity is associated with an increased risk of ischemic stroke.-   [17] Zheng X L. Annu Rev Med 2015 66: 211-225, ADAMTS-13 and von    Willebrand factor in thrombotic thrombocytopenic purpura.-   [18] E P 2172544.-   [19] Thomas M R, de Groot R, Scully M A, Crawley J T. EBioMedicine    2015 2, 942-952. Pathogenicity of Anti-ADAMTS-13 Autoantibodies in    Acquired Thrombotic Thrombocytopenic Purpura.-   [20] Denorme F, Langhauser F, Desender L, Vandenbulcke A,    Rottensteiner H, Plaimauer B, François O, Andersson T, Deckmyn H,    Scheiflinger F, Kleinschnitz C, Vanhoorelbeke K, De Meyer S F. Blood    2016 127:2337-2345. ADAMTS-13-mediated thrombolysis of    t-PA-resistant occlusions in ischemic stroke in mice.-   [21] Diaz J A, Hawley A E, Alvarado C M, Berguer A M, Baker N K,    Wrobleski S K, Wakefield T W, Lucchesi B R, Myers D D Jr. Thromb    Haemost 2010 104 366-375. Thrombogenesis with continuous blood flow    in the inferior vena cava. A novel mouse model.-   [22] Feys, H B, Vandeputte, N, Palla R, Peyvandi F, Peerlinck K,    Deckmyn H, Lijnen H R and Vanhoorelbeke K. Jr. Thromb Haemost 2010    8: 2053-62. Inactivation of ADAMTS13 by plasmin as a potential cause    of thrombotic thrombocytopenic purpura

Acknowledgement of the above references herein is not to be inferred asmeaning that these are in any way relevant to the patentability of thepresently disclosed subject matter.

BACKGROUND OF THE INVENTION

Dysregulation of hemostasis is central to the pathogenesis of thromboticdisorders. Under physiologic conditions, fibrin formation andfibrinolysis are coordinately regulated both temporally and locally. Theendothelium contributes to maintaining this balance, in part, byreleasing von Willebrand Factor (vWF) multimers from specializedgranules in response to endothelial stress or damage, and by thatinitiates thrombus formation. vWF is a large and heterogeneous,multidomain adhesive glycoprotein (GP) that is essential for normalhemostatic function. It is synthesized in endothelial cells as a monomerthat dimerizes in the endoplasmic reticulum through a C-terminaldisulfide bond. Heterogeneous vWF multimers are stored withinWeibel-Palade bodies, from which they can be released constitutively andupon demand.

vWF multimers undergo shear-induced conformational changes formingultralarge multimers (ULvWF) along the luminal surface of the damagedendothelium that promote platelet adhesion that is followed by fibrinaccumulation and ends by thrombus formation. ULvWF activity isconstrained by proteolytic inactivation by a disintegrin andmetalloproteinase with a thrombospondin type 1 motif, member 13(ADAMTS-13) [1, 2]. ADAMTS-13 is deficient in patients with congenitalor acquired autoimmune thrombotic thrombocytopenic purpura (TTP) whodevelop disseminated microvascular thrombosis and have increasedtendency to develop Myocardial Infarction (MI), acute ischemic stroke(AIS) [3-7] and Deep vein thrombosis (DVT), where it is considered aspotential therapeutic target [8]. Most TTP patients have acquiredanti-ADAMTS-13 autoantibodies that inhibit enzyme function and/or clearit from the circulation. In one of these patients, inactivation ofADAMTS-13 by excessive proteolysis mediated via plasmin was reported[22].

ADAMTS-13 is a 190 kDa glycosylated metalloproteinase produced byendothelial and hepatic stellate cells. Cleavage of ULvWF multimers byADAMTS-13 reduces platelet adhesion and down-regulates thrombusformation. In vivo studies using a DVT model in mice, show that theformed clots contain substantial amount of vWF [8]. Likewise, recentstudies show that human cerebrovascular thrombi contain variable amountsof vWF [9, 10] and that multimers of vWF integrate into polymerizedfibrin [11] which correlate with resistance to tPA [12] but not toADAMTS-13 [9].

Previous experimental and clinical studies suggest that levels of vWFand ADAMTS-13 are linked to the risk of AIS, DVT and pulmonary emboli(PE), and that ADAMTS-13 also protects the brain from reperfusion injuryin AIS [13-17].

EP2172544 [18] discloses several ADAMTS-13 mutants exhibiting enhancedor reduced catalytic activity, created to provide ADAMTS-13 variantsthat display reduced immunogenicity. Specifically, this publicationteaches that such mutants may be produced by substituting a chargedamino acid such as arginine (e.g., R312 or R326 or R370), Lysine (e.g.,K318 or K608), glutamic acid (E), aspartic acid (D)) in thedisintegrin-like domain, the cysteine-rich domain or the spacer domain,with a different amino acid, especially an uncharged amino acid.Accordingly, this publication teaches that charged amino acid residuesof ADAMTS-13 such as arginine or lysine, must be replaced by unchargedamino acid residues, such as alanine.

Thrombotic and Thromboembolic events such as venous thromboembolism(VTE) including deep venous thrombosis (DVT), pulmonary emboli (PE) andacute ischemic stroke (AIS), are a major cause of death anddisabilities. There is currently no effective medical treatment of suchconditions, and therefore an unmet need exists for development ofeffective modulators for thrombotic processes.

SUMMARY OF THE INVENTION

In a first aspect, the invention relates to a mutant of a disintegrinand metalloproteinase with a thrombospondin type 1 motif, member 13(ADAMTS-13) that carries at least one mutation. The ADAMTS-13 mutantdisclosed herein displays resistance and/or reduced sensitivity tocleavage and/or inactivation by at least one tissue plasminogenactivator (tPA), or any mutant or variant thereof. In some embodiments,at least one of the mutations of the mutants of the invention maysubstitute the Arginine in position 312 (Arg312) or in any amino acidresidue adjacent to the Arg312 of the wild type ADAMTS-13 with a chargedamino acid residue, or any truncated variant thereof. It should beunderstood that residue 312, as indicated herein refers to the aminoacid sequence of the wild type ADAMTS-13, that may comprise the aminoacid sequence as denoted by SEQ ID NO: 2, or any variants or derivativesthereof.

In yet some other aspect, the invention relates to a compositioncomprising an effective amount of at least one ADAMTS-13 mutant, or anytruncated variant thereof. In some embodiments, the mutant may carry amutation and displays resistance and/or reduced sensitivity to cleavageand/or inactivation by at least one tPA, or any mutant or variantthereof. In some embodiments, the mutant carries at least one mutationthat substitute the Arg312 residue and/or any amino acid residueadjacent to the Arg312 of the wild type ADAMTS-13 with a charged aminoacid residue. It should be noted that the wild type ADAMTS-13 comprisesthe amino acid sequence as denoted by SEQ ID NO: 2, and therefore theposition 312, refers to SEQ ID NO: 2. In some embodiments, thecomposition may optionally further comprise at least onepharmaceutically acceptable carrier, diluent, excipient and/or additive.

In another aspect, the invention relates to a combined compositioncomprising a combination of at least one ADAMTS-13 mutant and/or anyvariant thereof, and at least one tPA or any functional fragments orvariants thereof. In some embodiments, the mutant may carry at least onemutation and displays resistance and/or reduced sensitivity to cleavageand/or inactivation by at least one tPA, or any mutant or variantthereof. In some embodiments, at least one of the mutations of themutants may substitute the Arg312 residue or any amino acid residueadjacent to the Arg312 of the wild type ADAMTS-13 with a charged aminoacid residue. It should be noted that the wild type ADAMTS-13 maycomprise the amino acid sequence as denoted by SEQ ID NO: 2, or anyvariants or derivatives thereof. Still further, in some embodiments, thecomposition may optionally further comprise at least onepharmaceutically acceptable carrier, diluent, excipient and/or additive.

In a further aspect, the invention relates to a method for thetreatment, amelioration, inhibition or prophylaxis of a disease,disorder, or condition associated with coagulation in a subject in needthereof. In some embodiments, the method may comprise the step ofadministering to the subject a therapeutically effective amount of atleast one ADAMTS-13 mutant, any truncated variant thereof, or anycomposition or combined composition comprising the mutant of theinvention. In certain embodiments, the mutants of the invention maycarry at least one mutation and display resistance and/or reducedsensitivity to cleavage and/or inactivation by at least one tPA, or anymutant or variant thereof. In some embodiments, at least one of themutations of the disclosed mutants may substitute the Arg312 residue orany amino acid residue adjacent to the Arg312 of the wild type ADAMTS-13with a charged amino acid residue. It should be noted that the wild typeADAMTS-13 may comprise the amino acid sequence as denoted by SEQ ID NO:2.

In another aspect, the invention relates to a method for the treatment,amelioration, inhibition or prophylaxis of a disease, disorder, orcondition associated with coagulation in a subject in need thereof,comprising the step of administering to a subject treated with at leastone tPA, or any mutant or variant thereof, a therapeutically effectiveamount of at least one ADAMTS-13 mutant, and/or any variant thereof orany composition comprising the mutant. In some embodiments, the mutantmay carry at least one mutation and display resistance and/or reducedsensitivity to cleavage and/or inactivation by at least one tPA, or anymutant or variant thereof. In some embodiments, the mutant carries atleast one mutation that substitutes the Arg312 residue or any amino acidresidue adjacent to said Arg312 of the wild type ADAMTS-13 with acharged amino acid residue. The wild type ADAMTS-13 may comprise theamino acid sequence as denoted by SEQ ID NO: 2, or any variants orderivatives thereof.

In yet another aspect, the invention relates to a kit comprising:

First (a), at least one ADAMTS-13 mutant, and/or any variant thereof, orany composition thereof. In certain embodiment, the mutant may carry atleast one mutation, and display resistance and/or reduced sensitivity tocleavage and/or inactivation by at least one tPA, or any mutant orvariant thereof. In some embodiments, the mutant carries at least onemutation that substitutes the Arg312 residue and/or any amino acidresidue adjacent to the Arg312 of the wild type ADAMTS-13 with a chargedamino acid residue. The said wild type ADAMTS-13 may comprise the aminoacid sequence as denoted by SEQ ID NO: 2. The second component (b), maycomprise at least one tPA or any functional fragments or variantsthereof, or any composition thereof.

In a further aspect, the invention relates to at least one ADAMTS-13mutant, and/or any variant thereof or any composition comprising themutant, for use in a method of treatment, amelioration, inhibition orprophylaxis of a disease, disorder, or condition associated withcoagulation in a subject in need thereof. In some embodiments, themutant may carry at least one mutation and displays resistance and/orreduced sensitivity to cleavage and/or inactivation by at least one tPA,or any mutant or variant thereof. In some embodiments, the mutantcarries at least one mutation that substitutes the Arg312 residue or anyamino acid residue adjacent to the Arg312 of the wild type ADAMTS-13with a charged amino acid residue. It should be noted that the wild typeADAMTS-13 comprises the amino acid sequence as denoted by SEQ ID NO: 2.

The invention further provides a therapeutically effective amount of atleast one ADAMTS-13 mutant and/or any variant thereof or any compositioncomprising the mutant for use in a method for the treatment,amelioration, inhibition or prophylaxis of a disease, disorder, orcondition associated with coagulation in a subject in need thereof. Itshould be noted that the subject is a subject being treated with atleast one tPA, or any mutant or variant thereof. In yet some furtherspecific embodiments, the mutant carries at least one mutation, anddisplays resistance and/or reduced sensitivity to cleavage and/orinactivation by at least one tPA, or any mutant or variant thereof. Inmore specific embodiments, the mutation carried by the disclosed mutantand/or variant substitutes the Arg312 residue or any amino acid residueadjacent to said Arg312 of the wild type ADAMTS-13 with a charged aminoacid residue.

These and other aspects of the invention will become apparent by thehand of the following description.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to better understand the subject matter that is disclosedherein and to exemplify how it may be carried out in practice,embodiments will now be described, by way of non-limiting example only,with reference to the accompanying drawings, in which:

FIG. 1A-C: tPA cleaves native ADAMTS-13 in cells and in human plasma

FIG. 1A: Western blot analysis of lysates from fetal liver cells (A549cells) incubated in saline (Lysates), with tPA (100 nM) (tPA) or tPA(100 nM) and aprotinin (1 μM) (tPA+Ap) for 2 hours. Blot was incubatedwith monoclonal anti-ADAMTS-13 antibody EPR6132 (Abcam).

FIG. 1B: Western blot analysis of human plasma incubated in saline(Plasma), with tPA (100 nM) (tPA) or tPA (100 nM) and aprotinin (1 μM)(tPA+Ap) for 2 hours. Blot was incubated with monoclonal anti-ADAMTS-13antibody EPR6132 (Abcam).

FIG. 1C: Graph showing percent of activity of ADAMTS-13 in A549 celllysates and human plasma. ADAMTS-13 activity was determined by technozymADAMTS-13 activity (Technoclone).

FIG. 2 : tPA inactivates ADAMTS-13 in vivo

Graph showing percent of activity of ADAMTS-13 and vWF in plasma from WTand tPA−/− mice. ADAMTS-13 activity was determined by technozymADAMTS-13 activity (Technoclone), vWF antigen (Ag) was measured by Vonwillebrand factor Ag hemosil reagent (instrumentation laboratory) andvWF Ristocetin cofactor hemosil reagent (instrumentation laboratory).

FIG. 3A-3B: ADAMTS-13 structure

FIG. 3A: Schematic representation of human ADAMTS-13, a multidomainprotein with Metalloprotease domain (MP), Disinterring-like domain(Disin), 8 Thrombospondin type-1 domains (1-8), Cysteine-rich domain(Cys), Spacer domain (S) and 2 CUB domains.

FIG. 3B: Schematic representation of truncated ADAMTS-13 variant (MDTCS)i.e. trADAMTS-13 that contains MP domain and is catalytically active.

FIG. 4A-4D: Characterization of the truncated ADAMTS-13 variant FIG. 4A:SDS-PAGE of the truncated ADAMTS-13 variant (trADAMTS-13) (Lanes 2-3).

FIG. 4B: Graph showing activity of ADAMTS-13 WT and trADAMTS-13 withoutor with preincubation with tPA using a technozym kit to measureADAMTS-13 activity (Technoclone).

FIG. 4C: Western blot analysis of truncated recombinant human ADAMTS-13(trADAMTS-13) (500 nM) incubated with tPA (100 nM) for 0, 1, 2, 4 and 8hours using monoclonal anti-ADAMTS-13 antibody (Abcam).

FIG. 4D: Western blot analysis using anti-vWF antibody (Abcam) of vWFmultimers in human plasma incubated in the presence or absence oftrADAMTS-13 (300 nM) alone (for 3 hours) or with preincubation with tPA(100 nM, for 2 hours) at 37° C.

FIG. 5 : Delineation of the trADAMTS-13 cleavage site SDS-PAGE gel (10%)of trADAMTS-13 incubated without or with tPA for 4 hours. Individualbands were excised and their amino acid sequences were determined.

FIG. 6A-6B: Mutations of the trADAMTS-13 cleavage site and generation oftPA resistant variants

FIG. 6A: SDS-PAGE of the truncated ADAMTS-13 variants: trADAMTS-13 WT(Lane 1), trADAMTS-13^(R312K) (Lane 2), trADAMTS-13^(R312A) (Lane 3),trADAMTS-13^(V313A)(Lane 4) and trADAMTS-13^(V313D) (Lane 5).

FIG. 6B: Graph showing catalytic activity of the truncated ADAMTS-13variants on vWF.

FIG. 7A-7B: trADAMTS-13^(R312K) is resistant to tPA cleavage FIG. 7A:Western blot analysis of trADAMTS-13 WT and trADAMTS-13^(R312K)incubated with tPA for the indicated periods of times. Blot wasincubated with monoclonal anti-ADAMTS-13 antibody (Abcam).

FIG. 7B: Graph showing percentage of ADAMTS-13 proteolytic activity onvWF of both ADAMTS-13 truncated proteins, trADAMTS-13 WT (Black) andtrADAMTS-13^(R312K)(Gray) determined after the indicated periods of timeof incubation with tPA.

FIG. 8 : trADAMTS-13 variants are resistant to cleavage by tPA Graphshowing residual activity of the trADAMTS-13 variants incubated with orwithout tPA for 6 hours. Proteolytic activity on vWF of the variants wasdetermined after the incubation. Residual activity shows the activityobtained in presence of tPA in comparison to the activity in the absenceof tPA.

FIG. 9A-9B: Effect of autoantibodies from TTP plasma on trADAMTS-13variants activity

FIG. 9A: Graph showing residual activity of trADAMTS-13 variantsincubated for 2 hours with plasma from patients with acquired ADAMTS-13deficiency. Proteolytic activity on vWF of the variants was determinedafter the incubation. Residual activity shows the activity obtainedafter incubation with TTP plasma in comparison to that detected beforethe incubation.

FIG. 9B: Graph showing activity of trADAMTS-13 WT andtrADAMTS-13^(R312K) incubated for 2 hours or 24 hours with plasma frompatients with acquired ADAMTS-13 deficiency. Proteolytic activity on vWFof the variants was determined before and after the incubation withplasma. The activity obtained after incubation with TTP plasma incomparison to that detected before the incubation (mean±SD, p<0.05, n=5each group).

FIG. 10 : Thrombolytic effect of trADAMTS-13 variants on VWF-richthrombus Graph showing time to reperfusion following no treatment(control), treatment with tPA (tPA), treatment with tPA andtrADAMTS-13^(WT) (tPA+trADAMTS-13^(WT)) treatment with tPA andtrADAMTS-13^(R312K) (tPA+trADAMTS-13^(R312K)), treatment withtrADAMTS-13^(WT) or treatment with the mutant alone trADAMTS-13^(R312K).Five minutes after occlusion, mice were given intravenous injection oftPA (0.5 mg/kg) alone or together with trADAMTS-13^(WT) ortrADAMTS-13^(R312K) (5 mg/kg each), and blood reperfusion was monitoredfor 120 minutes. In the control group, no improvement was detected inblood flow even after 120 minutes of monitoring.

FIG. 11A-11C: In vivo anti-vWF activity of trADAMTS-13 variants FIG.11A: Graph showing percentage of ADAMTS-13 activity in plasma of WT andtPA −/− mice injected IP with trADAMTS-13 WT or trADAMTS-13^(R312K) (1mg, after two hours).

FIG. 11B: Graph showing concentration of vWF in plasma of WT and tPA −/−mice injected IP with trADAMTS-13 WT or trADAMTS-13^(R312K) (1 mg, aftertwo hours).

FIG. 11C: Graph showing percentage of vWF activity in plasma of WT miceinjected IP with trADAMTS-13 WT or trADAMTS-13^(R312K) (1 mg, after twohours).

FIG. 12A-12B: In vivo anti-thrombotic activity of trADAMTS-13 variants

FIG. 12A: Graph showing the weight of Thrombus (mg) in WT and tPA −/−mice after inducing venous clots formation using an IVC stasis model(Blood 2015 125(16): 2558-2567).

FIG. 12B: Graph showing the weight of Thrombus (mg) in WT miceadministered with IP injection of (1 mg) trADAMTS-13 WT ortrADAMTS-13^(R312K) one hour before inducing venous clots formationusing an IVC stasis model (Blood 2015 125(16): 2558-2567).

FIG. 13 : In vivo anti-coagulant activity of trADAMTS-13 variants Graphshowing bleeding time (min) of WT mice administered with IP injection of(1 mg) trADAMTS-13 WT or trADAMTS-13^(R312K) two hours before inducingbleeding using the tail cut model bleeding. Tails were cut as in (Blood.2019 Jan. 31; 133(5):481-493) and the bleeding times were determined asin (Blood. 2019 Jan. 31; 133(5):481-493).

FIG. 14A-14B: Soluble fibrin inhibits tPA-mediated inactivation ofADAMTS-13

FIG. 14A: Graph showing activity of the trADAMTS-13 incubated for 4hours in the presence/absence of tPA (100 nM) and/or fibrin (2 μM)(mean±SD, n=3 each group).

FIG. 14B: Graph showing activity of full length ADAMTS-13 in serum fromWT mice incubated for 4 hours in PBS (serum) or PBS containing tPA (100nM) or tPA and fibrin (0.1 mg/ml) (mean±SD, n=3 each group).

DETAILED DESCRIPTION OF THE INVENTION

Recent evidence indicates that ADAMTS-13 plays an important role inthrombotic and thromboembolic events [20, 21]. Currently, there is noeffective medical treatment related conditions such as venousthromboembolism (VTE) including deep venous thrombosis (DVT) andpulmonary emboli (PE) and acute ischemic stroke (AIS).

In the present application, improved ADAMTS-13 variants were developed,specifically ADAMTS-13 exhibiting enhanced activity, which representnovel thrombolytic and anticoagulant agents.

Therefore, in a first aspect, the invention relates to a mutant and/orvariant of a disintegrin and metalloproteinase with a thrombospondintype 1 motif, member 13 (ADAMTS-13) that carries at least one mutation.In yet some further embodiments, the mutants and/or variants disclosedherein display resistance and/or reduced sensitivity to cleavage and/orinactivation by at least one tissue plasminogen activator (tPA), or anymutant or variant thereof.

In some embodiments, the mutant and/or variant of the present disclosurecarries at least one mutation. In some embodiments, at least one of themutations in the mutants of the invention may substitute the Arginine inposition 312 (Arg312) and/or in any amino acid residue adjacent to theArg312 of the wild type ADAMTS-13 with a charged amino acid residue, orany truncated variant thereof. It should be understood that residue 312,as indicated herein refers to the amino acid sequence of the wild typeADAMTS-13, that may comprise the amino acid sequence as denoted by SEQID NO: 2, or any variants, homologues or derivatives thereof.

ADAMTS-13 (a disintegrin and metalloproteinase with a thrombospondintype 1 motif, member 13), also known as von Willebrand factor-cleavingprotease (VWFCP), and also referred to herein as ADAMTS-13, ADAMTS13,and ADAMTS, is a zinc-containing metalloprotease enzyme that cleaves vonWillebrand factor (vWf), a large protein involved in blood clotting. Itis secreted into the blood and degrades large vWf multimers, decreasingtheir activity. Genomicaly, ADAMTS-13 shares many properties with the 19member ADAMTS family, all of which are characterized by a proteasedomain (the part that performs the protein hydrolysis), an adjacentdisintegrin domain and one or more thrombospondin domains. ADAMTS-13 infact has eight thrombospondin domains. It has no hydrophobictransmembrane domain, and hence it is not anchored in the cell membrane.Human ADAMTS-13 is a multidomain protein with Metalloprotease domain(MP), Disinterring-like domain (Disin), 8 Thrombospondin type-1 domains(1-8), Cysteine-rich domain (Cys), Spacer domain (S) and 2 CUB domains.The metalloprotease domain contains the catalytic site of ADAMTS-13 thatcleaves vWF. The ADAMTS-13 gene maps to the ninth chromosome (9q34) andits accession number is NM_139025.4. It encodes for the amino acidsequence having the accession number NP_620594.1.

In some embodiments, the human wild type ADAMTS-13 protein may comprisethe amino acid sequence as denoted by SEQ ID NO: 2, or any variants,homologues or derivatives thereof. In some further embodiments, the wildtype ADAMTS-13 protein may be encoded by a nucleic acid sequencecomprising the nucleic acid sequence as denoted by SEQ ID NO: 1, or anyvariants, homologues or derivatives thereof.

The present invention provides mutants of the ADAMTS-13 protein. Theterm “mutant” or “mutant protein,” as used herein, refers to a proteinproduct encoded by a gene with mutation. Specifically, it should beappreciated that “ADAMTS-13 mutant” as used herein includes a mutatednative and recombinant mutated ADAMTS-13 protein, as well as modifiedforms of ADAMTS-13 that display increased activity in comparison withthe enzymatic activity displayed by the wild type ADAMTS-13. Morespecifically, the term “mutation” as herein defined refers to a changein the nucleotide sequence of the genome of an organism, for example, inthe nucleic acid sequence encoding the ADAMS-13 protein. Mutations mayor may not produce observable (phenotypic) changes in thecharacteristics of the encoded protein. However, in some embodiments,the mutants disclosed herein display a modified sensitivity toproteolytic cleavage and/or inactivation. Mutation can result in severaldifferent types of change in the DNA sequence; these changes may have noeffect, alter the product of a gene, or prevent the gene fromfunctioning properly or completely. There are generally three types ofmutations, namely single base substitutions, insertions and deletionsand mutations defined as “chromosomal mutations”. The term “single basesubstitutions” as herein defined refers to a single nucleotide basewhich is replaced by another. These single base changes are also calledpoint mutations. There are two types of base substitutions, namely,“transition” and “transversion”. When a purine base (i.e., Adenosine orThymine) replaces a purine base or a pyrimidine base (Cytosine, Guanine)replaces a pyrimidine base, the base substitution mutation is termed a“transition”. When a purine base replaces a pyrimidine base orvice-versa, the base substitution is called a “transversion”.

Single base substitutions may be further classified according to theireffect on the genome, as follows. In missense mutations the new basealters a codon, resulting in a different amino acid being incorporatedinto the protein chain.

In nonsense mutations the new base changes a codon that specified anamino acid into one of the stop codons (taa, tag, tga). This will causetranslation of the mRNA to stop prematurely and a truncated protein tobe produced. This truncated protein will be unlikely to functioncorrectly.

Mutation may also arise from insertions of nucleic acids into the DNA orfrom duplication or deletions of nucleic acids therefrom. As hereindefined, the term “insertions and deletions” refers to extra base pairsthat are added or deleted from an encoding nucleic acid sequence,respectively. Insertions and deletions of one or two bases or multiplesof one or two bases cause, inter alia, frame shift mutations (i.e.,these mutations shift the reading frame of the gene). In someembodiments, the mutant and/or variant of the present disclosure carriesat least one mutation that leads to “substitution”, e.g., replacement ofat least one amino acid residue with another residue, specifically,replacement of amino acid in position 312 of the ADAMTS-13 protein,and/or of any adjacent amino acid residue/s as discussed herein after,with any other amino acid residue.

Specifically, the invention relates to ADAMTS-13 mutant having amutation in the amino acid residue Arg312 or in at least one adjacentamino acid residue. As used herein, term “adjacent amino acid residue”refers to an amino acid being at a position of about 1 to 5 or moreresidues upstream or alternatively, downstream to residue 312 (alsoreferred to herein as minus or plus, respectively). Specifically, atleast one, at least two, at least three, at least four, at least five ormore residues upstream of residue 312, specifically, residues 307, 308,309, 310, 311. Or alternatively, at least one, at least two, at leastthree, at least four, at least five or more residues downstream ofresidue 312, specifically, residues 313, 314, 315, 316, 317. In morespecific embodiments, the mutants of the invention may carry asubstitution of residue 312, or of an amino acid residue located tworesidues plus/minus to residue 312. For example, in some embodiments,amino acid residue adjacent to Arg312 corresponds to amino acid residue310, 311, 313 or 314. In some specific embodiments, an adjacent aminoacid residue to Arginine 312 may be Valine 313. Thus, in someembodiments, the mutant/s and or variant/s disclosed herein may carry amutation that leads to substitution and/or replacement of amino acidresidue at position 312. In yet some further embodiments, the mutant ofthe present disclosure may carry at least one mutation that substitutesat least one residue adjacent to residue 312, for example, a mutationthat result in substitution of at least one of residues 307, 308, 309,310, 311, 313, 314, 315, 316, 317, with another amino acid residue.Still further, in some embodiments, the mutant and/or variant/sdisclosed herein may carry at least one mutation replacing amino acidresidues at position 312 and in addition, a substitution of at least oneof the following residues 307, 308, 309, 310, 311, 313, 314, 315, 316,317, of ADAMTS-13 protein, specifically as denoted by SEQ ID NO: 2. Inyet some specific embodiments, the mutant of the present invention maycarry at least one mutation that substitutes residues 312 and 313 ofADAMTS-13.

As indicated above, the ADAMTS-13 mutants of the invention comprisereplacement of residue 312 and/or any adjacent residue with any chargedamino acid residue. A charged amino acid residue may be residues thatare either negative (i.e. de-protonated) at physiological pH, forexample, aspartic acid (Asp, D) and glutamic acid (Glu, E), or positive(i.e. protonated) at physiological pH, for example, the lysine (Ly, K),arginine (Arg, R) and histidine (His, H).

In some embodiments, the charged amino acid residue of the replacingspecific amino acid residues in the ADAMTS-13 mutant or any truncatedvariant thereof may be any one of lysine (K), aspartic acid (D),glutamic acid (E) or histidine (H).

In some specific embodiments, the charged amino acid residue of theADAMTS-13 mutant or any truncated variant thereof may be lysine.

In some embodiments, the ADAMTS-13 mutant may carry a mutationsubstituting the Arginine in position 312 with any amino acid residue,with the proviso that said amino acid reside is not Alanine.

In yet some further specific embodiments, the ADAMTS-13 mutant of thepresent disclosure may carry a mutation substituting the Arginine inposition 312 to lysine. Such mutant is designated R312K. In someembodiments, the mutant may comprise the amino acid sequence as denotedby SEQ ID NO:11 or any variants, homologues or derivatives thereof.

In some embodiments, the present disclosure provides mutants or variantsof ADAMTS-13, and/or any variants of the disclosed mutants. Variants ofthe disclosed mutants may encompass any truncated, extended, modifiedvariants of the disclosed mutants. Further variants encompassed by thepresent disclosure are defined herein below.

Thus, in some embodiments, the further disclosure encompasses atruncated variant of the disclosed mutants. A truncated variant of agiven protein as used herein refers to any variant comprising an aminoacid sequence that was shortened, trimmed, cut or reduced in at leastone amino acid residue of the N-terminal end and/or the C-terminal endof the protein. More specifically, the variants encompassed by thepresent disclosure are truncated variants that are shortened in at leastabout 1 to about 800 or more residues, either in the C′- and/orN′-termini thereof, specifically, 1, 10, 20, 30, 40, 50, 60, 70, 80, 90,100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750,800 amino acid residues (aa's) or more. In some embodiments, theADAMTS-13 truncated variant in accordance with the present disclosure istruncated in the N and the C′ termini thereof. Still further, thetruncated variant of the mutants disclosed herein are truncated in theC-terminal end of the ADAMTS-13 molecule. In yet some furtherembodiments, the truncated mutants disclosed herein are truncated in atleast one of the following domains: at least one of the thrombospondintype 1 (TSP) repeats 2-8, and the two 2 C-terminal CUB domains (CUB).Still further, in some embodiments, the truncated variant of the presentdisclosure has a deletion or truncation of the C′ terminal CUB domain.In yet some further embodiments, the two C-terminal CUB domains aretruncated in the mutant. Still further, in some embodiments, thetruncated mutant is truncated in the two C-terminal CUB domains and TSPrepeat 8. In some further embodiments, the truncated mutant is truncatedin the two C-terminal CUB domains and TSP repeats 8 and 7. In somefurther embodiments, the truncated mutant is truncated in the twoC-terminal CUB domains and TSP repeats 8, 7 and 6. In some furtherembodiments, the truncated mutant is truncated in the two C-terminal CUBdomains and TSP repeats 8, 7, 6 and 5. In some further embodiments, thetruncated mutant is truncated in the two C-terminal CUB domains and TSPrepeats 8, 7, 6, 5 and 4. In some further embodiments, the truncatedmutant is truncated in the two C-terminal CUB domains and TSP repeats 8,7, 6, 5, 4 and 3. In some further embodiments, the truncated mutant istruncated in the two C-terminal CUB domains and TSP repeats 8, 7, 6, 5,4, 3 and 2. In some embodiments, the truncated mutant of the presentdisclosure comprises the following domains of ADAMTS-13, themetalloprotease (MP), Disintegrin-like domain (Dis), thrombospondin type1 (TSP) repeat (1) and cysteine-rich and spacer domain (Cys).

In yet some further particular and non-limiting embodiments, a truncatedvariant of the mutant of the invention may comprise the amino acidsequence as denoted by SEQ ID NO:5, or any variants or derivativesthereof. Accordingly, in some further embodiments, the truncatedADAMTS-13 mutant of the invention may be encoded by a nucleic acidsequence as denoted by SEQ ID NO:18, or any variants or derivativesthereof. Still further, as will be indicated in more detail hereinafter, truncated variants disclosed by the present disclosure include,but are not limited to any variants comprising the amino acid sequenceas denoted by any one of; SEQ ID NO: 7 (truncated WT), SEQ ID NO: 15(truncated ADAMTS-13^(R312A)), SEQ ID NO: 16 (truncatedADAMTS-13^(V313A)), SEQ ID NO: 17 (truncated V313D), any variants,derivatives thereof, and any combinations thereof.

Still further, it should be understood that the present disclosureencompasses any ADAMTS-13 mutant and/or variant, specifically anytruncated variants that comprise at least the MP catalytic domain of themolecule, and therefore retain the ability of cleaving vWF. Stillfurther, the mutants of the present disclosure or any variants thereofretain resistance and/or reduced sensitivity to cleavage and/orinactivation by at least one proteolytic protein, specifically, by tPA.

As indicated above, the mutants or variants of the present disclosuremay comprise at least one mutation in residue 312, and/or in anyadjacent residue. Thus, in some further embodiments, the adjacent aminoacid residue of the ADAMTS-13 mutant of the invention or any truncatedvariant thereof, may be valine 313. In such embodiments, the mutant maycarry a mutation substituting valine 313 with aspartic acid. In someembodiment, the mutant may be designated V313D. In yet some furtherembodiments, such mutant may comprise the amino acid sequence as denotedby SEQ ID NO: 14 or any derivative or variants thereof. In yet somefurther embodiments, a truncated version of such mutant may comprise theamino acid sequence as denoted by SEQ ID NO: 17, or any derivative orvariants thereof. Such truncated mutant may be encoded in someembodiments by the nucleic acid sequence as denoted by SEQ ID NO: 21. Inyet some specific and non-limiting embodiments, the mutants disclosedherein may comprise substitution of resides 312 and 313. Thus, in someembodiments, such double mutant may comprise R312K and V313D. In someembodiments, such double mutants may comprise the amino acid sequence asdenoted by SEQ ID NO: 23 and the truncated form thereof as denoted bySEQ ID NO; 24.

Still further, ADAMTS-13 mutants encompassed by the invention includethe R312A mutant (replacing Arg 312 with alanine), such mutant maycomprise the amino acid sequence as denoted by SEQ ID NO: 12 or anyderivative or variants thereof, and the truncated version thereof maycomprise the amino acid sequence as denoted by SEQ ID NO: 15 or anyderivative or variants thereof. It yet some further embodiments, thetruncated version of the R312A mutant may be encoded by a nucleic acidsequence comprising SEQ ID NO: 19.

In some further embodiments, the invention further encompasses ADAMTS-13mutant that carry a replacement of valine 313 with alanine V313A. Suchmutant may comprise the amino acid sequence as denoted by SEQ ID NO: 13or any derivative or variants thereof, and the truncated version thereofmay comprise the amino acid sequence as denoted by SEQ ID NO: 16 or anyderivative or variants thereof. It yet some further embodiments, thetruncated version of the R312A mutant may be encoded by a nucleic acidsequence comprising SEQ ID NO: 22.

In yet some further embodiments, the invention provides ADAMTS-13variants that are truncated variant comprising the amino acid sequenceas denoted by SEQ ID NO: 7 or any derivative or variants thereof,encoded by the nucleic acid sequence as denoted by SEQ ID NO: 6.

As indicated above, the invention provides ADAMTS-13 mutants andvariants thereof, specifically, as denoted by SEQ ID NO: 5, 11, 12, 13,14, 15, 16, 17, 23 and 24, or any variants or derivatives thereof. Asindicated above, all mutants and variants disclosed herein, arefunctional mutant and/or variant in the sense of they all retain theability of cleaving vWF, and/or they all retain resistance and/orreduced sensitivity to cleavage and/or inactivation by tPA. Stillfurther, in some embodiments, the mutants of the present disclosureretain ability of dissolving blood clots.

It should be appreciated that the invention encompasses any variant orderivative of the ADAMTS-13 mutant polypeptides of the invention and anypolypeptides that are substantially identical or homologue to thepolypeptides encoded by the nucleic acid sequence of the invention. Theterm “derivative” or “variant” is used to define amino acid sequences(polypeptide), with any insertions, deletions, substitutions andmodifications to the amino acid sequences (polypeptide) that do notalter the activity of the original polypeptides. Specifically, abilityof cleaving vWF, and/or resistance and/or reduced sensitivity tocleavage and/or inactivation by tPA. By the term “derivative” it is alsoreferred to homologues, variants and analogues thereof. Proteinsorthologs or homologues having a sequence homology or identity to theproteins of interest in accordance with the invention, specifically, allADAMTS-13 mutants and/or variants described herein, may share at least50%, at least 60% and specifically 65%, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher, specifically ascompared to the entire sequence of the proteins of interest inaccordance with the invention, for example, any of the proteins thatcomprise the amino acid sequence as denoted by any one of SEQ ID NO: 5,11, 12, 13, 14, 15, 16, 17, 23 and 24. Specifically, homologs thatcomprise or consists of an amino acid sequence that is identical in atleast 50%, at least 60% and specifically 65%, 70%, 75%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher to the entiresequence of the mutants and/or variants of the invention, specifically,any of the mutants that comprise the amino acid sequence as denoted bySEQ ID NO: 5, 11, 12, 13, 14, 15, 16, 17, 23, 24 and any derivatives,homologues and variants thereof.

In some embodiments, derivatives refer to polypeptides, which differfrom the polypeptides specifically defined in the present invention byinsertions, deletions or substitutions of amino acid residues. It shouldbe appreciated that by the terms “insertion/s”, “deletion/s” or“substitution/s”, as used herein it is meant any addition, deletion orreplacement, respectively, of amino acid residues to the polypeptidesdisclosed by the invention, of between 1 to 50 amino acid residues,between 20 to 1 amino acid residues, and specifically, between 1 to 10amino acid residues. More particularly, insertion/s, deletion/s orsubstitution/s may be of any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10amino acids. It should be noted that the insertion/s, deletion/s orsubstitution/s encompassed by the invention may occur in any position ofthe modified peptide, as well as in any of the N′ or C′ termini thereof.

With respect to amino acid sequences, one of skill will recognize thatindividual substitutions, deletions or additions to a nucleic acid,peptide, polypeptide, or protein sequence which alters, adds or deletesa single amino acid or a small percentage of amino acids in the encodedsequence is a “conservatively modified variant” where the alterationresults in the substitution of an amino acid with a chemically similaramino acid. Conservative substitution tables providing functionallysimilar amino acids are well known in the art. Such conservativelymodified variants are in addition to and do not exclude polymorphicvariants, interspecies homologues, and alleles of the invention. Forexample, substitutions may be made wherein an aliphatic amino acid (G,A, I, L, or V) is substituted with another member of the group, orsubstitution such as the substitution of one polar residue for another,such as arginine for lysine, glutamic for aspartic acid, or glutaminefor asparagine. Each of the following eight groups contains otherexemplary amino acids that are conservative substitutions for oneanother:

-   -   1) Alanine (A), Glycine (G);    -   2) Aspartic acid (D), Glutamic acid (E);    -   3) Asparagine (N), Glutamine (Q);    -   4) Arginine (R), Lysine (K);    -   5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);    -   6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);    -   7) Serine (S), Threonine (T); and    -   8) Cysteine (C), Methionine (M).

Thus, in some embodiments, the present disclosure encompasses thespecified polypeptides, or any derivatives thereof, specifically aderivative that comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or moreconservative substitutions to the amino acid sequences as denoted by anyone of SEQ ID NO: 5, 11, 12, 13, 14, 15, 16, 17, 23 24. Morespecifically, amino acid “substitutions” are the result of replacing oneamino acid with another amino acid having similar structural and/orchemical properties, i.e., conservative amino acid replacements. Aminoacid substitutions may be made on the basis of similarity in polarity,charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues involved. For example, nonpolar“hydrophobic” amino acids are selected from the group consisting ofValine (V), Isoleucine (I), Leucine (L), Methionine (M), Phenylalanine(F), Tryptophan (W), Cysteine (C), Alanine (A), Tyrosine (Y), Histidine(H), Threonine (T), Serine (S), Proline (P), Glycine (G), Arginine (R)and Lysine (K); “polar” amino acids are selected from the groupconsisting of Arginine (R), Lysine (K), Aspartic acid (D), Glutamic acid(E), Asparagine (N), Glutamine (Q); “positively charged” amino acids areselected form the group consisting of Arginine (R), Lysine (K) andHistidine (H) and wherein “acidic” amino acids are selected from thegroup consisting of Aspartic acid (D), Asparagine (N), Glutamic acid (E)and Glutamine (Q). Variants of the polypeptides of the invention mayhave at least 80% sequence similarity or identity, often at least 85%sequence similarity or identity, 90% sequence similarity or identity, orat least 95%, 96%, 97%, 98%, or 99% sequence similarity or identity atthe amino acid level, with the entire protein of interest, such as thevarious polypeptides of the invention.

As shown in Examples 2 and 3, the inventors have first demonstrated thattPA cleaves the ADAMTS-13 protein and inhibits its activity. The exactcleavage site was determined to be at R³¹²-V³¹³. Several mutantsperturbing the R312V313 bond in ADAMTS-13 were created, some of themexhibiting resistance to cleavage to tPA, as demonstrated in Example 4.Therefore, in some embodiments, as discussed herein above, the ADAMTS-13mutant of the invention or any truncated variant thereof displayresistance, and/or reduced sensitivity to cleavage by at least oneserine protease.

In certain embodiment, the serine protease may be at least one of tissueplasminogen activator (tPA), urokinase plasminogen activator (uPA),plasmin, thrombin or granulocyte elastase, or other plasminogenactivators like streptokinase or any functional fragments or variantsthereof.

In some specific embodiments, the ADAMTS-13 mutant of the invention orany truncated variant thereof may display resistance and/or reducedsensitivity to cleavage and/or inactivation by at least one tissueplasminogen activator (tPA), or any mutant or variant thereof.

It should be appreciated that the term tPA used herein for the tissueplasminogen activator (also known as PLAT; enzyme entry EC 3.4.21.68),relates to a secreted serine protease that converts and activates theproenzyme plasminogen to a potent fibrinolytic enzyme plasmin. tPA issynthesized in vascular endothelial cells as a single polypeptide chainthat undergoes proteolytic cleavage by plasmin or trypsin at a centrallylocated arginine-isoleucine bond, resulting in a 2-chaindisulfide-linked form composed of the N-terminally derived heavy chainand the C-terminal light chain. The tPA gene (DNA acc. NT_167187.1mapped to chr. 8p11.21) contains 14 exons encoding the heavy chaindomain including two kringle regions (K1 and K2) and regions homologousto growth factors and the light chain domain comprising the serineprotease catalytic site. Alternative splicing of the tPA gene results inmultiple transcript variants encoding different isoforms taking part inmultiple biological processes, apart from fibrinolysis, such as cellmigration and tissue remodeling. Increased tPA activity causeshyperfibrinolysis manifested as excessive bleeding; decreased tPAactivity leads to hypofibrinolysis which can result in thrombosis orembolism. tPA linked phenotypes include familial hyperfibrinolysis (dueto increased tPA release) and familial thrombophilia (due to decreasedtPA release (OMIM num. 612348). It should be noted that in someembodiments, tPA, as used herein refers to the human tPA that comprisethe amino acid sequence encoded by the nucleic acid sequence comprisingthe sequence as denoted by SEQ ID NO: 3, or any variants or derivativesthereof. In some further embodiments, the human tPA may comprise anamino acid sequence encoded by a nucleic acid sequence comprising asequence having at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or 100% homology with the sequence asdenoted by SEQ ID NO: 3. In yet some further embodiments, such human tPAmolecule may comprise the amino acid sequence as denoted by SEQ ID NO:4. In yet some other embodiments, the human tPA may comprise an aminoacid sequence having at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology with the sequence asdenoted by SEQ ID NO: 4, and any variants, homologues and derivativesthereof.

As indicated herein, the disclosed mutants and variants displayresistance to cleavage, specifically, proteolytic cleavage (e.g.,proteolysis) by at least one serine protease such as tPA. Proteolysis,as used herein is the breakdown of proteins into smaller polypeptides oramino acids. In some embodiments, resistance and/or reduced sensitivityto proteolytic cleavage as used herein is meant that the ADAMTS-13mutant disclosed herein is less vulnerable, and/or displays reducedvulnerability and/or sensitivity and/or increased resistance toproteolytic cleavage and/or inactivation by at least about 5% to 100% ormore. In some embodiments, reduced sensitivity and/or vulnerability ismeant that about 5% to 100% of the mutated ADAMTS-13 molecule is notcleaved and/or inactivated by tPA. More specifically, in someembodiments, the ADAMTS-13 mutant of the present disclosure displayreduced sensitivity to cleavage and/or inactivation by tPA,specifically, a reduced sensitivity of at least 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,100%, 200%, 300%, 400%, 500%, 600%, 70%, 800%, 900%, 1000% or more ascompared to the sensitivity of the wild type ADAMTS-13. Still further,the mutant of the present disclosure display resistance or increasedresistance to cleavage, degradation and/or inactivation by tPA,specifically, an increase of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%,400%, 500%, 600%, 70%, 800%, 900%, 1000% or more resistance to cleavage,or alternatively, reduced sensitivity as compared to the wild typeADAMTS-13. Inactivation, as referred to herein, is meant any reductionof about 5% to about 100%, in the disclosed activity of ADAMTS-13, e.g.,proteolysis of any substrate, specifically, the vWF, speed and extent ofdissolving blood clots and restoring blood flow, as compared to the wildtype ADMTS-13. Still further, in some embodiments, about 5% to 100% ofthe mutated ADAMTS-13 of the present disclosure in a given compositionor any biological fluid (e.g., blood, plasma, either in a subject or invitro), is not cleaved and/or inactivated by tPA present in the samebiological fluid.

Still further, in some embodiments, the ADAMTS-13 mutant and/or variantprovided herein, or any truncated variant thereof, display an increasedor enhanced activity, as compared to the WT ADAMTS-13. In yet some otherembodiments, the ADAMTS-13 mutant of the invention may exhibit enhancedenzymatic activity. In some specific embodiment, the enzymatic activityof the ADAMTS-13 mutant of the invention may refer to cleavage of uWF.According to some embodiments, wherein indicated “increased” or“enhanced” activity, it is meant that such increase or enhancement maybe an increase or elevation of between about 10% to 100% of theADAMTS-13 mutant activity in comparison with the Wild type ADAMTS-13protein. The terms “increased”, “augmented” and “enhanced” as usedherein relate to the act of becoming progressively greater in size,amount, number, or intensity. Particularly, an increase of 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 100%, 200%, 300%, 400%, 500%, 600%, 70%, 800%, 900%, 1000% ormore of the activity as compared to a suitable control, e.g., the Wildtype ADAMTS-13 protein. It should be further noted that increase orelevation may be also an increase of about 2 to 10⁶ folds or more. Withregards to the above, it is to be understood that, where provided,percentage values such as, for example, 10%, 50%, 120%, 500%, etc., areinterchangeable with “fold change” values, i.e., 0.1, 0.5, 1.2, 5, etc.,respectively. Therefore, the term increase refers to an increase ofabout 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100,200, 300, 400, 500, 600, 700, 800, 900, 1000 folds or more.

As noted above, the ADAMTS-13 mutants of the invention display enhancedcleavage of vWF. von Willebrand factor (VWF) is a blood glycoproteininvolved in hemostasis. VWF is a large multimeric glycoprotein presentin blood plasma and produced constitutively as ultra-large VWF inendothelium (in the Weibel-Palade bodies), megakaryocytes (α-granules ofplatelets), and subendothelial connective tissue. The basic VWF monomeris a 2050-amino acid protein. Every monomer contains a number ofspecific domains with a specific function; elements of note include:

-   -   the D′/D3 domain, which binds to factor VIII (von Willebrand        factor type D domain);    -   the A1 domain, which binds to: platelet GPIb-receptor, heparin        and possibly collagen;    -   the A2 domain, which must partially unfold to expose the buried        cleavage site for the specific ADAMTS-13 protease that        inactivates VWF by making much smaller multimers (the partial        unfolding is affected by shear flow in the blood, by calcium        binding, and by the lump of a sequence-adjacent “vicinal        disulfide” at the A2-domain C-terminus);    -   the A3 domain, which binds to collagen (von Willebrand factor        type A domain);    -   the C4 domain, in which the RGD motif binds to platelet integrin        αIIbβ3 when this is activated (von Willebrand factor type C        domain);    -   the other C domains, which may interact in ER dimers: the larger        protein show six beads of (C and C-like) domains under cryo-EM;    -   the “cystine knot” domain (at the C-terminal end of the        protein), which VWF shares with platelet-derived growth factor        (PDGF), transforming growth factor-β (TGFβ) and β-human        chorionic gonadotropin (PHCG, of pregnancy test fame), (von        Willebrand factor type C domain);

Monomers are subsequently N-glycosylated, arranged into dimers in theendoplasmic reticulum and into multimers in the Golgi apparatus bycrosslinking of cysteine residues via disulfide bonds. With respect tothe glycosylation, VWF is one of only a few proteins that carry ABOblood group system antigens. vWFs coming out of the Golgi are packagedinto storage organelles, Weibel-Palade bodies (WPBs) in endothelialcells and α-granules in platelets.

Multimers of VWF can be extremely large, >20,000 kDa, and consist ofover 80 subunits of 250 kDa each. Only the large multimers arefunctional.

In yet some further alternative and/or additional embodiments, increasedactivity when referred to the ADAMTS-13 mutant/s and/or variant/sdisclosed herein, may relate to the ability of the mutant/s and/orvariant/s of the present disclosure to reduce size and/or volume and/orweight of at least one blood clot, in about 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%,or more, as compared with the ability of the wild type ADAMTS-13 toreduce size and/or volume and/or weight of at least one blood clot.Enhanced activity (two folds or more) of the mutant of the presentinvention to dissolve blood clot is shown by FIG. 12 . Still further, insome embodiments enhanced activity of the disclosed ADAMTS-13 mutant/sand/or variant/s may refer to reduced time of dissolving blood clots,and/or time of restoring blood flow, as also clearly exemplified by FIG.10 and Example 6. More specifically, in some embodiments, “time ofrestoring blood flow” as used herein, is meant the time (e.g., minutes)required for recovery from a reduction to about 25% or less (and evencessation) of the blood flow (e.g., the blood volume passing over timethrough a blood vessel). In some embodiments, the reduction in bloodflow is occurred for example by occlusive thrombus or blood clot. Stillfurther, the time required to restore the blood flow to about 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% and 100% ofthe blood flow before occlusion or blockage occurred.

In some embodiments, the ADAMTS-13 mutants disclosed herein and anycompositions thereof significantly reduce the time required forrestoring the blood flow and/or dissolving the blood clot, whenadministered to a subject.

A thrombus, colloquially called a blood clot, as used herein, is thefinal product of blood coagulation. The two major components to athrombus are aggregated platelets and red blood cells that form a plug,and a mesh of cross-linked fibrin protein. Thrombi are classified intotwo major groups depending on their location and relative amount ofplatelets and red blood cells. More specifically, arterial or whitethrombi (characterized by predominance of platelets), and venous or redthrombi (characterized by predominance of red blood cells). An occlusivethrombus is a blockage that completely seals off a blood vessel, where anon-occlusive thrombus only partially blocks off a blood vessel (e.g.,vein or artery).

As shown by FIG. 7 and Example 4, the mutant/s and/or variant/sdisclosed herein, specifically, the ADAMTS-13^(R312K) mutant, whencompared to the WT ADAMTS-13, displayed extended duration of proteolyticactivity (e.g., 8 hr to 24 hr vs 2 hr to 6 hr) as reflected byproteolysis of vWF. These results indicate increased stability andactivity over time, as defined by a prolonged half-life of the disclosedmutants and are clearly valuable for therapeutic applications. Indeed,as disclosed in Example 4, the half-life of the disclosed mutanttrADAMTS-13^(R312K), is about 344.6+/−131.1 min, whereas the half-lifeof the wild-type molecule is much lower (132.8+/−41.3 min). In someembodiment, the half-life of the mutant trADAMTS-13^(R312K) is betweenabout 150 to about 750 minutes or more, specifically, about 150, 160,170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300,310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440,450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580,590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720,730, 740, 750 minutes or more.

Thus, in yet some further embodiments, the ADAMTS-13 mutant and/orvariant disclosed herein or any truncated variant thereof, display aprolonged half-life relative to ADAMTS-13 wild type. In someembodiments, the prolonged half-life of the ADAMTS-13 mutant disclosedherein extends the duration of the ADAMTS activity, as reflected byproteolysis of vWF, up to 24 hr or more, specifically, up to 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24 hrs or more. The mutants of the invention therefore display in someembodiments an extended activity window. A biological half-life (alsoknown as elimination half-life, pharmacologic half-life), also denotedby the abbreviation of a biological substance such as the ADAMTS-13mutant of the present disclosure, is the time it takes to reduce themaximum concentration (C_(max)) to half of its maximum concentration inthe blood plasma in about 1% to about 100% or more. More specifically,the ADAMTS-13 mutants of the present disclosure display a half-life thatis prolonged in about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%,600%, 70%, 800%, 900%, 1000% or more. It is to be understood that, whereprovided, percentage values such as, for example, 10%, 50%, 120%, 500%,etc., are interchangeable with “fold change” values, i.e., 0.1, 0.5,1.2, 5, etc., respectively. Therefore, the term prolonged refers tohalf-life increased in about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50,60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 foldsor more.

Still Further, additional beneficial effects of the ADAMTS-13 mutants ofthe invention relate to resistance to anti ADAMTS-13 autoantibodiesfound for example in plasma of Thrombotic Thrombocytopenic Purpura (TTP)patients, as shown in Example 5. Therefore, in some embodiments, theADAMTS-13 mutant of the invention may exhibit resistance to antiADAMTS-13 autoantibodies.

It should be understood that the various properties of the disclosedADAMTS-13 mutants as discussed herein are applicable for any of theADAMTS-13 mutant/s and/or variant/s disclosed in any of the aspects ofthe present disclosure.

The invention provides mutants of ADAMTS 13 protein. The terms “protein”or “polypeptide” as used herein refers to amino acid residues, connectedby peptide bonds. A polypeptide sequence is generally reported from theN-terminal end containing free amino group to the C-terminal endcontaining free carboxyl group and may include any polymeric chain ofamino acids. In some embodiments, a polypeptide has an amino acidsequence that occurs in nature. In some embodiments, a polypeptide hasan amino acid sequence that does not occur in nature. In someembodiments, a polypeptide has an amino acid sequence that containsportions that occur in nature separately from one another (i.e., fromtwo or more different organisms, for example, human and non-humanportions). In some embodiments, a polypeptide has an amino acid sequencethat is engineered in that it is designed and/or produced through actionof the hand of man. More specifically, “Amino acid sequence” or “peptidesequence” is the order in which amino acid residues connected by peptidebonds, lie in the chain in peptides and proteins. Amino acid sequence isoften called peptide, protein sequence if it represents the primarystructure of a protein, however one must discern between the terms“Amino acid sequence” or “peptide sequence” and “protein”, since aprotein is defined as an amino acid sequence folded into a specificthree-dimensional configuration and that had typically undergonepost-translational modifications, such as phosphorylation, acetylation,glycosylation, manosylation, amidation, carboxylation, sulfhydryl bondformation, cleavage and the like.

As indicated herein, the present disclosure provides various ADAMTS-13mutants and/or variant. It should be appreciated however, that theinvention further encompasses any nucleic acid sequence encoding themutants and/or variant disclosed herein. The term “nucleic acid”,“nucleic acid sequence”, or “polynucleotide” and “nucleic acid molecule”refers to polymers of nucleotides, and includes but is not limited todeoxyribonucleic acid (DNA), ribonucleic acid (RNA), DNA/RNA hybridsincluding polynucleotide chains of regularly and/or irregularlyalternating deoxyribosyl moieties and ribosyl moieties (i.e., whereinalternate nucleotide units have an —OH, then and —H, then an —OH, thenan —H, and so on at the 2′ position of a sugar moiety), andmodifications of these kinds of polynucleotides, wherein the attachmentof various entities or moieties to the nucleotide units at any positionare included. The terms should also be understood to include, asequivalents, analogs of either RNA or DNA made from nucleotide analogs.Preparation of nucleic acids is well known in the art. Moreover, thepresent disclosure further provides any vector, expression vector or anyconstruct comprising at least one nucleic acid molecule encoding theclaimed mutants or variants. Vectors, as used herein, are nucleic acidmolecules of particular sequence that can be introduced into a hostcell, thereby producing a transformed host cell. A vector may includenucleic acid sequences that permit it to replicate in a host cell, suchas an origin of replication. A vector may also include one or moreselectable marker genes and other genetic elements known in the art,including promoter elements that direct nucleic acid expression. Manyvectors, e.g., plasmids, cosmids, minicircles, phage, viruses, (asdetailed below) useful for transferring nucleic acids into target cellsmay be applicable in the present invention. The vectors comprising thenucleic acid(s) may be maintained episomally, e.g., as plasmids,minicircle DNAs, viruses such cytomegalovirus, adenovirus, or they maybe integrated into the target cell genome, through homologousrecombination or random integration.

Still further, in some embodiments, the preset disclosure provides anyhost cell expressing any nucleic acid sequence that encodes theADAMTS-13 mutants and variant/s disclosed herein.

The term “host cell” includes a cell into which a heterologous (e.g.,exogenous) nucleic acid or protein has been introduced. Persons of skillupon reading this disclosure will understand that such terms refer notonly to the particular subject cell, but also is used to refer to theprogeny of such a cell, as well as any population of cells comprisingthe host cell/s of the invention. Because certain modifications mayoccur in succeeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term “host cell”.The term “host cells” as used herein refers to any cell known to askilled person wherein the ADAMTS-13 variant and/or mutated molecule orany functional fragments or peptides thereof or any nucleic acidmolecule according to the invention may be introduced. For example, ahost cell may be eukaryotic or prokaryotic cell of a unicellular ormulti-cellular organism. More specifically, a host cell may include, butis not limited to a yeast, fungi, an insect cell, an invertebrate cell,vertebrate cell, mammalian cell and the like.

It should be understood that in some embodiments, the ADAMTS-13 mutantsof the present disclosure may be produced either recombinantly, forexample by expressing the encoding nucleic acid sequence or any vectorthereof, in any appropriate host cell, for example, any of the hostcells disclosed herein.

In yet some other aspect, the invention relates to a compositioncomprising an effective amount of at least one ADAMTS-13 mutant, or anytruncated variant thereof. In some embodiments, the mutant/s and/orvariants of the composition disclosed herein may display resistanceand/or reduced sensitivity to cleavage and/or inactivation by at leastone seine protease, for example, tPA.

In some embodiments, the mutant may carry a mutation and at least one ofthe mutation/s may substitute the Arg312 residue and/or any amino acidresidue adjacent to the Arg312 of the wild type ADAMTS-13 with a chargedamino acid residue. It should be noted that the wild type ADAMTS-13comprises the amino acid sequence as denoted by SEQ ID NO: 2, andtherefore the position 312, refers to SEQ ID NO: 2. In some embodiments,the composition may optionally further comprise at least onepharmaceutically acceptable carrier, diluent, excipient and/or additive.

In some other embodiments, the compositions of the invention or anycombined compositions may comprise any of the ADAMTS-13 mutantsdisclosed by the invention, as specifically described above.

In some further embodiments, the composition of the invention mayfurther comprise a therapeutically effective amount of at least oneserine protease. In some alternative embodiments, the serine proteasemay be at least one of tPA, uPA, plasmin, thrombin or granulocyteelastase, or other plasminogen activators like streptokinase or anyfunctional fragments or variants thereof.

In some specific embodiments, the composition of the invention mayfurther comprise a therapeutically effective amount of at least onetissue plasminogen activator (tPA), or any mutant or variant thereof.

“Pharmaceutically or therapeutically acceptable carrier” refers to acarrier medium which does not interfere with the effectiveness of thebiological activity of the active ingredients. As mentioned herein, thecompositions provided by the invention optionally further comprise atleast one pharmaceutically acceptable excipient or carrier. As usedherein “pharmaceutically acceptable carrier” includes any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents and the like. The use of such media and agents for pharmaceuticalactive substances is well known in the art. Except as any conventionalmedia or agent is incompatible with the active ingredient, its use inthe therapeutic composition is contemplated.

As used herein “pharmaceutically acceptable carrier/diluents/excipient”includes any and all solvents, dispersion media, coatings and the like.The use of such media and agents for pharmaceutical active substances iswell known in the art. Except as any conventional media or agent isincompatible with the active ingredient, its use in the therapeuticcomposition is contemplated.

Pharmaceutical compositions used to treat subjects in need thereofaccording to the invention generally comprise a buffering agent, anagent who adjusts the osmolarity thereof, and optionally, one or morepharmaceutically acceptable carriers, excipients and/or additives asknown in the art. Supplementary active ingredients can also beincorporated into the compositions. The carrier can be solvent ordispersion medium containing, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyethylene glycol, andthe like), suitable mixtures thereof, and vegetable oils. The properfluidity can be maintained, for example, by the use of a coating, suchas lecithin, by the maintenance of the required particle size in thecase of dispersion and by the use of surfactants.

In various embodiments, the final solution of any of the compositions ofthe invention may be adjusted with a pharmacologically acceptable acid,base or buffer. In some embodiments, the compositions of the inventionmay be suitable for systemic administration. The pharmaceuticalcomposition of the invention can be administered and dosed by themethods of the invention, in accordance with good medical practice. Morespecifically, the compositions used in the methods and kits of theinvention, described herein after, may be adapted for administration bysystemic, parenteral, intraperitoneal, transdermal, oral (includingbuccal or sublingual), rectal, topical (including buccal or sublingual),vaginal, intranasal and any other appropriate routes. Such formulationsmay be prepared by any method known in the art of pharmacy, for exampleby bringing into association the active ingredient with the carrier(s)or excipient(s).

The phrases “systemic administration”, “administered systemically” asused herein mean the administration of a compound, drug or othermaterial other than directly into the central blood system, such that itenters the patient's system and, thus, is subject to metabolism andother like processes. The phrases “parenteral administration” and“administered parenterally” as used herein means modes of administrationother than enteral and topical administration, usually by injection, andincludes, without limitation, intravenous, intramuscular, intraarterial,intrathecal, intracapsular, intraorbital, intracardiac, intradermal,intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticulare, subcapsular, subarachnoid, intraspinal and intrasternalinjection and infusion.

Systemic administration includes parenteral injection by intravenousbolus injection, by intravenous infusion, by sub-cutaneous,intramuscular, intraperitoneal injections or by suppositories, bypatches, or by any other clinically accepted method, including tablets,pills, lozenges, pastilles, capsules, drinkable preparations, ointment,cream, paste, encapsulated gel, patches, boluses, or sprayable aerosolor vapors containing these complexes and combinations thereof, whenapplied in an acceptable carrier. Alternatively, to any pulmonarydelivery as by oral inhalation such as by using liquid nebulizers,aerosol-based metered dose inhalers (MDI's), or dry powder dispersiondevices.

In other embodiments the pharmaceutical composition may be adapted fortopical administration. By “topical administration” it is meant that thepharmaceutical composition and the carrier may be adapted to any mode oftopical administration including: epicutaneous, transdermal, oral,bronchoalveolar lavage, ophtalmic administration, enema, nasaladministration, administration to the ear, administration by inhalation.

Regardless of the route of administration selected, the compositions ofthe present invention, which may be used in a suitable hydrated form,and/or the pharmaceutical compositions of the present invention, areformulated into pharmaceutically-acceptable dosage forms by conventionalmethods known to those of skill in the art.

Pharmaceutical compositions used to treat subjects in need thereofaccording to the invention generally comprise a buffering agent, anagent who adjusts the osmolarity thereof, and optionally, one or morepharmaceutically acceptable carriers, excipients and/or additives asknown in the art. Supplementary active ingredients can also beincorporated into the compositions. The carrier can be solvent ordispersion medium containing, for example, water, ethanol, and suitablemixtures thereof. The proper fluidity can be maintained, for example, bythe use of a coating, such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants.

The fact that the ADAMTS-13 mutants of the invention display resistanceto cleavage and/or inactivity by tPA, is of particular interest forcombined composition suitable for conditions that necessitate thecleavage by both enzymes. Indeed, as shown, also in Example 6, theADAMTS-13 mutants developed by the inventors exhibit a synergisticactivity with tPA on cleavage vWF-rich clots. As indicated herein, theADAMTS-13 mutants of the invention, as well as in the compositions,combined compositions and kits of the invention, and used by the methodsdescribed herein after, may act in synergy with tPA. Therefore, in somefurther embodiments, the ADAMTS-13 mutant of the invention may exhibitsynergistic activity with tPA.

Synergy, as used herein refer to the interaction or cooperation of twoor more substances, compounds, or any other agents, specifically, atleast one ADAMTS-13 mutants of the invention and tPA or any functionalfragments or variants thereof, to produce a combined effect greater thanthe sum of their separate effects.

Thus, in another aspect, the invention relates to a combined compositioncomprising a combination of at least one ADAMTS-13 mutant or anytruncated variant thereof, and at least one tPA or any functionalfragments or variants thereof. In some embodiments, the mutant may carryat least one mutation. In some embodiments, the mutant/s and/or variantsof the combined composition display resistance and/or reducedsensitivity to cleavage and/or inactivation by at least one serineprotease, specifically, tPA.

In yet some further embodiments, the combined composition of the presentdisclosure comprises at least one ADAMTS-13 mutant and/or variant thatcarry at least one mutation that substitute the Arg312 residue and/orany amino acid residue adjacent to said Arg312 of the wild typeADAMTS-13 with a charged amino acid residue. It should be noted that thewild type ADAMTS-13 may comprise the amino acid sequence as denoted bySEQ ID NO: 2, or any variants or derivatives thereof. Still further, insome embodiments, the composition may optionally further comprise atleast one pharmaceutically acceptable carrier, diluent, excipient and/oradditive.

In some embodiments, the mutant comprised in the combined composition ofthe invention may be any of the ADAMTS-13 mutants of the invention,specifically, any of the mutants defined above, or any combinationsthereof. It should be understood that the tPA comprised within thecombined composition disclosed herein is in some embodiments, the tPA asdisclosed herein above in connection with other aspects of theinvention.

In some further embodiments, the combined composition of the inventionmay further or alternatively, comprise a therapeutically effectiveamount of at least one additional or alternative serine protease. Insome alternative embodiments, the serine protease may be at least one ofuPA, plasmin, thrombin or granulocyte elastase, or other plasminogenactivators like streptokinase or any functional fragments or variantsthereof.

Still further, as shown by FIG. 14 and Example 8, the presence of fibrinled to reduced cleavage and/or inactivation of ADAMTS-13 by tPA andtherefore enhanced the activity of ADAMTS-13 on vWF. Thus, in somespecific embodiments, the combined composition may further comprise inaddition to at least one ADAMTS-13 mutant/s and/or variant/s, and tPA,also fibrin.

Still further, in yet some further embodiments, the present disclosurefurther encompasses compositions comprising wild type ADAMTS-13, tPA andfibrin, and further encompasses any use of this combined composition, asdiscussed in the present disclosure.

The anti-thrombotic activity of the ADAMTS-13 mutants was alsodemonstrated in vivo in mice, as shown in Example 7. Mice injected withthe ADAMTS-13R312K mutant retained greater capacity to proteolyze vWFand an increased effect on bleeding was observed as well, indicatingthat the mutants of the invention may be used as potent anti-coagulationagents.

Thus, in yet some other embodiments, the mutant/s and/or variant/s ofthe present disclosure, as well as any composition or combinedcomposition thereof, may be particularly suitable for use in a methodfor the treatment, amelioration, inhibition or prophylaxis of a disease,disorder, or condition associated with coagulation in a subject in needthereof. Coagulation, also known as clotting, is the process by whichblood changes from a liquid to a gel, forming a blood clot. Itpotentially results in hemostasis, the cessation of blood loss from adamaged vessel, followed by repair. The mechanism of coagulationinvolves activation, adhesion and aggregation of platelets, as well asdeposition and maturation of fibrin. Coagulation begins almost instantlyafter an injury to the blood vessel has damaged the endothelium liningthe blood vessel. Exposure of blood to the subendothelial spaceinitiates two processes: changes in platelets, and the exposure ofsubendothelial tissue factor to plasma Factor VII, which ultimatelyleads to cross-linked fibrin formation. Platelets immediately form aplug at the site of injury; this is called primary hemostasis. Secondaryhemostasis occurs simultaneously: additional coagulation (clotting)factors beyond Factor VII (listed below) respond in a cascade to formfibrin strands, which strengthen the platelet plug.

Disorders of coagulation are disease states which can result inbleeding—hemorrhage or bruising—or obstructive clotting—thrombosis. Asused herein, the term “disease, disorder, or condition associated withcoagulation” refers to any condition and/or disorder that relatesdirectly and/or indirectly to obstructive clotting, i.e., excessiveunregulated clothing and coagulation process, or alternatively oradditionally, to any condition caused by a blood clot located in a bloodvessel affecting particular organ, that may lead to ischemic condition.This term will be further defined herein after in connection withadditional aspects of the present disclosure.

As discussed above, the in vivo effect of the mutants or variants of thepresent disclosure on resolving blood clots is applicable in treatingany condition associated directly or indirectly with coagulation.

Thus, in a further aspect, the invention relates to methods for thetreatment, amelioration, inhibition or prophylaxis of a disease,disorder, or condition associated with coagulation in a subject in needthereof. In some embodiments, the method may comprise the step ofadministering to the subject a therapeutically effective amount of atleast one ADAMTS-13 mutant/s and/or variant (e.g., any truncated variantthereof), or any composition or combined composition comprising themutant/s and/or variant/s of the invention. In certain embodiments, themutants of the invention may carry at least one mutation. In certainembodiments, the mutant/s and/or variant/s used by the methods disclosedherein may display resistance and/or reduced sensitivity to cleavageand/or inactivation by at least one serine protease, for example, tPA.In yet some further specific embodiments, the mutant/s used by thedisclosed method may carry at least one mutation that substitute theArg312 residue and/or any amino acid residue adjacent to the Arg312 ofthe wild type ADAMTS-13 with a charged amino acid residue. It should benoted that the wild type ADAMTS-13 may comprise the amino acid sequenceas denoted by SEQ ID NO: 2.

In some embodiments, the charged amino acid residue of the mutant/sand/or variant/s suitable for the method of the invention may be any oneof lysine, aspartic acid, glutamic acid or histidine.

In some specific embodiments, the charged amino acid residue of themutant/s and/or variant/s suitable for the method of the invention maybe lysine.

In some more specific embodiments, mutant/s and or variant/s suitablefor the methods of the invention may carry a mutation substituting theArginine in position 312 to lysine and may be designated R312K, orADAMTS-13R³¹²K. In certain embodiment, the mutant may comprise the aminoacid sequence as denoted by SEQ ID NO:11, or any variants or derivativesthereof. In yet some further embodiments, such variants or derivativesmay be the truncated variant of the mutant. In some specificembodiments, such truncated variant may comprise the amino acid sequenceas denoted by SEQ ID NO:5, or any variant or derivatives thereof. Itshould be understood that ADAMTS-13 mutant/s and/or variant/s suitablefor the present disclosure may be any of the mutant/s and/or variant/sdisclosed by the present disclosure in connection with other aspects ofthe invention.

In some other specific embodiments, any mutant/s or variant/s ofADAMTS-13 disclosed herein is suitable for the method of the invention,with the proviso that the mutant and/or variant does not carry amutation substituting the Arginine in position 312 to Alanine. In somespecific embodiments such mutant may comprise the amino acid sequence asdenoted by SEQ ID NO: 12, or the truncated version thereof that comprisethe amino acid sequence as denoted by SEQ ID NO: 15. Thus, according tosome specific embodiments, the methods disclosed herein may use andtherefore administer any of the ADAMTS-13 mutant/s and/or variant/sdisclosed herein, provided that such mutants are not the mutants of SEQID NO: 5 or of SEQ ID NO: 12.

Still further, in some other embodiments, the adjacent amino acidresidue of the mutant suitable for the methods of the invention may bevaline 313. In some specific embodiments, such mutant/s that aresuitable in the present disclosure may carry a mutation substitutingvaline 313 with aspartic acid. In some embodiments such mutant maycomprise the amino acid sequence as denoted by SEQ ID NO: 14, or thetruncated version thereof that comprise the amino acid sequence asdenoted by SEQ ID NO: 17.

In some further embodiments, the mutant suitable for the method of theinvention may be any of the ADAMTS-13 mutants of the invention,specifically, any of the mutants defined above.

As indicated above, in some other embodiments, the ADAMTS-13 mutant/sand/or variant/s suitable for the method of the invention may displayresistance and/or decreased sensitivity to cleavage and/or inactivationby at least one serine protease. In certain embodiments, the serineprotease may be at least one of tPA, and/or any other serine protease,for example, uPA, plasmin, thrombin or granulocyte elastase or otherplasminogen activators like streptokinase or any functional fragments orvariants thereof, specifically, tPA.

Thus, in certain embodiments, the methods of the invention may furthercomprise the step of administering to the subject, a therapeuticallyeffective amount of at least one tPA or any functional fragments orvariants thereof or any composition thereof.

In some embodiments, the mutant/s and/or variant/s used by the methodsdisclosed herein (e.g., any truncated variant) display an increasedactivity. It should be understood that an increased activity of thedisclosed mutants/and/or variant/s as used herein is as defined inconnection with previous aspects of the present disclosure (e.g.,proteolysis of vWF, resolving blood clots, recovering blood low, andmore).

In yet some further embodiments, the ADAMTS-13 mutant/s or any variant/sthereof (e.g., the truncated variant) used by the methods disclosedherein display a prolonged half-life relative to ADAMTS-13 wild type.

In some embodiments, the methods disclosed herein are particularlysuitable for treating disease, disorder, or condition that relatesand/or is associated directly or indirectly to coagulation. The normalbalance between clot formation and breakdown can be changed by thepresence of certain genetic or acquired defects leading to abnormal clotformation. It should be therefore understood that the term“coagulation-related disorders” and/or “disorders and/or conditionsassociated with coagulation” as used herein, encompass any conditionassociated directly of indirectly with abnormal clot formation and/ormaintenance. Reasons for the clot formation and breakdown processes tobe unbalanced toward abnormal clot formation include blood vesselinjury, venous stasis (lack of movement of the blood in the veins), andclotting disorders. These three factors make up Virchow's triad. Analteration in any one of these three factors can lead to abnormalclotting. All risk factors for DVT or PE fall into one of these threecategories. A venous thromboembolic event (VTE) is either a DVT or PE orboth in the same patient. Clotting disorders are present in the majorityof patients who have a DVT. D-dimer is a by-product of clot breakdownand is elevated in DVT or PE conditions.

There are two types of clotting disorders. The first is a hereditarydisorder and the second is an acquired disorder. The hereditary clottingdisorders come are divided in two groups: group 1 is characterized withlack of anti-clotting factors in the blood, while group 2 ischaracterized with an increased amount of pro-clotting factors in theblood.

Group 1 disorders include anti-thrombin deficiency, protein Cdeficiency, and protein S deficiency. Group 2 disorders includeactivated protein C resistance (Factor V Leiden mutation), prothrombinG20210A mutation, and elevated levels of Factors VIII, IX, and XI. Ingeneral, the Group 1 disorders are less common but more likely to causeabnormal clotting than Group 2 disorders. It should be appreciated thatthe methods disclosed herein may be applicable for any coagulationdisorder, either hereditary or acquired of group 1 and/or group 2, inany stage or degree of any of these disorders.

In some specific and non-limiting embodiments, the therapeutic methodsdisclosed herein may be applicable for at least one of deep venousthrombosis (DVT), pulmonary emboli (PE), acute ischemic stroke (AIS),acute myocardial function (AMI), thrombotic thrombocytopenic purpura(TTP), disseminated intravascular coagulation (DIC), hemolytic-uremicsyndrome (HUS), cerebral infarction or systemic lupus erythematosus(SLE).

In some embodiments, the methods disclosed herein may be applicable forDVT. As used herein, the term “deep venous thrombosis (DVT)” refers tothe formation of a blood clot in a deep vein, most commonly the legs.Symptoms may include pain, swelling, redness, or warmth of the affectedarea. It should be appreciated that about half of cases have nosymptoms. Complications may include pulmonary embolism (PE), as a resultof detachment of a clot which travels to the lungs, and post-thromboticsyndrome.

Risk factors for such condition include recent surgery, cancer, trauma,lack of movement, obesity, smoking, hormonal birth control, pregnancyand the period following birth, antiphospholipid syndrome, and certaingenetic conditions. Genetic factors include deficiencies ofantithrombin, protein C, and protein S, and factor V Leidenmutation. Theunderlying mechanism typically involves some combination of decreasedblood flow rate, increased tendency to clot, and injury to the bloodvessel wall.

Individuals suspected of having DVT may be assessed using a clinicalprediction rule such as the Wells score. A D-dimer test may also be usedto assist with excluding the diagnosis or to signal a need for furthertesting. Diagnosis is most commonly confirmed by ultrasound of thesuspected veins. Together, DVT and pulmonary embolism are known asvenous thromboembolism (VTE).

Anticoagulation (blood thinners) is the standard treatment. Typicalmedications include low-molecular-weight heparin, warfarin, or a directoral anticoagulant.

Still further, in some embodiments, the methods disclosed herein may beapplicable for Pulmonary embolism (PE). PE is a blockage of an artery inthe lungs by a substance that has moved from elsewhere in the bodythrough the bloodstream (embolism). Symptoms of a PE may includeshortness of breath, chest pain particularly upon breathing in, andcoughing up blood. Symptoms of a blood clot in the leg may also bepresent, such as a red, warm, swollen, and painful leg. Signs of a PEinclude low blood oxygen levels, rapid breathing, rapid heart rate, andsometimes a mild fever. Severe cases can lead to passing out, abnormallylow blood pressure, and sudden death.

PE usually results from a blood clot in the leg that travels to thelung. The risk of blood clots is increased by cancer, prolonged bedrest, smoking, stroke, certain genetic conditions, estrogen-basedmedication, pregnancy, obesity, and after some types of surgery. A smallproportion of cases are due to the embolization of air, fat, or amnioticfluid. Diagnosis is based on signs and symptoms in combination with testresults. If the risk is low, a blood test known as a D-dimermay rule outthe condition. Otherwise, a CT pulmonary angiography, lungventilation/perfusion scan, or ultrasound of the legs may confirm thediagnosis.

In some embodiments, the methods of the invention may be applicable forAIS. Acute ischemic stroke (AIS) occurs when there is a sudden occlusionof the arterial blood supply to part of the brain and is most commonlymanifested by focal neurological deficits.

In an ischemic stroke, blood supply to part of the brain is decreased,leading to dysfunction of the brain tissue in that area. There areseveral major reasons for stroke, including, thrombosis (obstruction ofa blood vessel by a blood clot forming locally), embolism (obstructiondue to an embolus from elsewhere in the body), systemic hypoperfusion(general decrease in blood supply, e.g., in shock) and cerebral venoussinus thrombosis.

A stroke without an obvious explanation is termed cryptogenic (ofunknown origin), and constitutes 30-40% of all ischemic strokes.

In some embodiments, the methods of the invention may be applicable foracute ischemic stroke. There are various classification systems foracute ischemic stroke. The Oxford Community Stroke Projectclassification (OCSP, also known as the Bamford or Oxfordclassification) relies primarily on the initial symptoms; based on theextent of the symptoms, the stroke episode is classified as totalanterior circulation infarct (TACI), partial anterior circulationinfarct (PACI), lacunar infarct (LACI) or posterior circulation infarct(POCI). These four entities predict the extent of the stroke, the areaof the brain that is affected, the underlying cause, and the prognosis.The TOAST (Trial of Org 10172 in Acute Stroke Treatment) classificationis based on clinical symptoms as well as results of furtherinvestigations; on this basis, a stroke is classified as being due to(1) thrombosis or embolism due to atherosclerosis of a large artery, (2)an embolism originating in the heart, (3) complete blockage of a smallblood vessel, (4) other determined cause, (5) undetermined cause (twopossible causes, no cause identified, or incomplete investigation).Users of stimulants, such as cocaine and methamphetamine are at a highrisk for ischemic strokes.

In some embodiments, the methods of the invention may be applicable forAMI. Acute Myocardial Infarction (AMI) refers to tissue death(infarction) of the heart muscle (myocardium). It is a type of acutecoronary syndrome, which describes a sudden or short-term change insymptoms related to blood flow to the heart. Unlike other causes ofacute coronary syndromes, such as unstable angina, a myocardialinfarction occurs when there is cell death, as measured by a blood testfor biomarkers (the cardiac protein troponin or the cardiac enzymeCK-MB). When there is evidence of an MI, it may be classified as an STelevation myocardial infarction (STEMI) or non-ST elevation myocardialinfarction (NSTEMI) based on the results of an ECG.

The phrase “heart attack” is often used non-specifically to refer to amyocardial infarction and to sudden cardiac death. An MI is differentfrom—but can cause—cardiac arrest, where the heart is not contracting atall or so poorly that all vital organs cease to function, thus causingdeath. It is also distinct from heart failure, in which the pumpingaction of the heart is impaired. However, an MI may lead to heartfailure.

Chest pain is the most common symptom of acute myocardial infarction andis often described as a sensation of tightness, pressure, or squeezing.Levine's sign, in which a person localizes the chest pain by clenchingone or both fists over their sternum, has classically been thought to bepredictive of cardiac chest pain, although a prospective observationalstudy showed it had a poor positive predictive value.

Chest pain may be accompanied by sweating, nausea or vomiting, andfainting, and these symptoms may also occur without any pain at all. Inwomen, the most common symptoms of myocardial infarction includeshortness of breath, weakness, and fatigue.

In yet some further embodiments, the methods of the invention may beapplicable for TTP. Thrombotic thrombocytopenic purpura (TTP) is a blooddisorder that results in blood clots forming in small blood vesselsthroughout the body. This results in a low platelet count, low red bloodcells due to their breakdown, and often kidneys, heart, and braindysfunction. Symptoms may include large bruises, fever, weakness,shortness of breath, confusion, and headache. Repeated episodes mayoccur.

In about half of cases a trigger is identified, while in the remainderthe cause remains unknown. Known triggers include bacterial infections,certain medications, autoimmune diseases such as lupus, and pregnancy.The underlying mechanism typically involves antibodies inhibiting theenzymeADAMTS-13. This results in decreased break down of large multimersof von Willebrand factor (vWF) into smaller units. Less commonly TTP isinherited from a person's parents, known as Upshaw-Schulman syndrome,such that ADAMTS-13 dysfunction is present from birth. Diagnosis istypically based on symptoms and blood tests. It may be supported bymeasuring activity of or antibodies against ADAMTS-13. With plasmaexchange the risk of death has decreased from more than 90% to less than20%. Immunosuppressants, such as glucocorticoids, and rituximabmay alsobe used.

Still further, in some embodiments, the methods of the invention may beapplicable for DIC. Disseminated intravascular coagulation (DIC) is acondition in which blood clots form throughout the body, blocking smallblood vessels. Symptoms may include chest pain, shortness of breath, legpain, problems speaking, or problems moving parts of the body. Asclotting factors and platelets are used up, bleeding may occur. This mayinclude blood in the urine, blood in the stool, or bleeding into theskin. Complications may include organ failure.

Relatively common causes include sepsis, surgery, major trauma, cancer,and complications of pregnancy. Less common causes include snake bites,frostbite, and burns. There are two main types of DIC, acute (rapidonset) and chronic (slow onset). Diagnosis is typically based on bloodtests. Findings may include low platelets, low fibrinogen, high INR, orhigh D-dimer. Treatment is mainly directed towards the underlyingcondition. Other measures may include giving platelets, cryoprecipitate,or fresh frozen plasma. Heparin may be useful in the slowly developingform.

In some further embodiments, the methods of the invention may beapplicable for HUS. Hemolytic-uremic syndrome (HUS) is a group of blooddisorders characterized by low red blood cells, acute kidney failure,and low platelets. Initial symptoms typically include bloody diarrhea,fever, vomiting, and weakness. Kidney problems and low platelets thenoccur as the diarrhea is improving. While children are more commonlyaffected, adults may have worse outcomes. Complications may includeneurological problems and heart failure.

Most cases occur after infectious diarrhea due to a specific type of E.coli called O157:H7. Other causes include S. pneumoniae, Shigella,Salmonella, and certain medications. The underlying mechanism typicallyinvolves the production of Shiga toxin by the bacteria. Atypicalhemolytic uremic syndrome (aHUS) is due to a genetic mutation andpresents differently. Though both cause widespread inflammation andmultiple blood clots in small blood vessels, a condition known asthrombotic microangiopathy.

Treatment involves supportive care and may include dialysis, steroids,blood transfusions, or plasmapheresis.

The early symptoms can include diarrhea (which is often bloody), stomachcramps, mild fever, or vomiting that results in dehydration and reducedurine. Related symptoms and signs include lethargy, decreased urineoutput, blood in the urine, kidney failure, low platelets, (which areneeded for blood clotting), and destruction of red blood cells(microangiopathic hemolytic anemia). High blood pressure, jaundice (ayellow tinge in skin and the whites of the eyes), seizures, and bleedinginto the skin can also occur.

Still further, in some embodiments, the methods disclosed herein may beapplicable for cerebral infraction. A cerebral infarction is an area ofnecrotic tissue in the brain resulting from a blockage or narrowing inthe arteries supplying blood and oxygen to the brain. The restrictedoxygen due to the restricted blood supply causes an ischemic stroke thatcan result in an infarction, if the blood flow is not restored within arelatively short period of time. The blockage can be due to a thrombus,an embolus or an atheromatous stenosis of one or more arteries. Whicharteries are problematic will determine which areas of the brain areaffected (infarcted). These varying infarcts will produce differentsymptoms and outcomes.

Symptoms of cerebral infarction are determined by the parts of the brainaffected. If the infarct is located in primary motor cortex,contralateral hemiparesis is said to occur. With brainstem localization,brainstem syndromes are typical: Wallenberg's syndrome, Weber'ssyndrome, Millard-Gubler syndrome, Benedikt syndrome or others.Infarctions will result in weakness and loss of sensation on theopposite side of the body. Physical examination of the head area willreveal abnormal pupil dilation, light reaction and lack of eye movementon opposite side. If the infarction occurs on the left side brain,speech will be slurred. Reflexes may be aggravated as well.

Still further, in some embodiments, the methods of the invention may beapplicable for SLE. Systemic lupus erythematosus (SLE), also knownsimply as lupus, is an autoimmune disease in which the body's immunesystem mistakenly attacks healthy tissue in many parts of the body.Symptoms vary between people and may be mild to severe. Common symptomsinclude painful and swollen joints, fever, chest pain, hair loss, mouthulcers, swollen lymph nodes, feeling tired, and a red rash which is mostcommonly on the face. Often there are periods of illness, called flares,and periods of remission during which there are few symptoms.

The cause of SLE is not clear. It is thought to involve geneticstogether with environmental factors. Among identical twins, if one isaffected there is a 24% chance the other one will be as well. Female sexhormones, sunlight, smoking, vitamin D deficiency, and certaininfections, are also believed to increase the risk. The mechanisminvolves an immune response by autoantibodies against a person's owntissues. These are most commonly anti-nuclear antibodies and they resultin inflammation. Diagnosis can be difficult and is based on acombination of symptoms and laboratory tests. There are a number ofother kinds of lupus erythematosus including discoid lupuserythematosus, neonatal lupus, and subacute cutaneous lupuserythematosus. There is no cure for SLE. Treatments may include NSAIDs,corticosteroids, immunosuppressants, hydroxychloroquine, andmetotrexate. SLE significantly increases the risk of cardiovasculardisease with this being the most common cause of death.

It should be understood that the preset disclosure, and specifically anyof the methods and compositions disclosed herein are applicable for anyof the disclosed coagulation-associated disorders as well as to anystage and grade of each of these conditions and symptoms thereof.

In some embodiments, the methods disclosed herein may further comprisethe step of administering to the subject, a therapeutically effectiveamount of at least one tPA or any functional fragments or variantsthereof or any composition thereof.

As discussed herein before and as exemplified by the present Examples,to further enhance the thrombolytic effect of the disclosed ADAMTS-13mutants and variants, the present disclosure further provides a combinedtherapeutic approach, combining the use of fibrinolytic compounds suchas tPA with the disclosed ADAMTS-13 variants and mutants. Thus, in someembodiments, the therapeutic and/or prophylactic methods disclosedherein may further comprise the step of administering to the treatedsubject, a therapeutically effective amount of at least one tPA or anyfunctional fragments or variants thereof or any composition thereof.Alternatively, the combined treatment may be performed using a combinedcomposition comprising both fibrinolytic compounds. Thus, in somefurther embodiments, the methods of the invention may comprise the stepof administering to the subject, a therapeutically effective amount of acombined composition comprising a combination of at least one ADAMTS-13mutant and/or any truncated variant thereof; and at least one tPA or anyfunctional fragments or variants thereof. In some embodiments, thecombined composition may be as defined above. Therefore, the inventionfurther encompasses the option of combined therapy.

It should be further appreciated and understood that the mutants,compositions and methods disclosed herein may be combined with any otheranti coagulating agent, when used for treating and/or preventingcoagulation related conditions. The therapeutic anti-coagulating mutantsmay therefore enhance any anti-coagulating effect of any otheranticoagulating agent such as heparin, Warfarin (Comadin), clexane andthe like.

In another aspect, the invention relates to a method for the treatment,amelioration, inhibition or prophylaxis of a disease, disorder, orcondition associated with coagulation in a subject in need thereof. Insome embodiments, the methods disclosed herein may comprise the step ofadministering to a subject treated with at least one tissue plasminogenactivator (tPA), or any mutant or variant thereof, any/or with anyfibrinolytic compounds, a therapeutically effective amount of at leastone ADAMTS-13 mutant/s and/or variant/s (e.g., any truncated variantthereof) or any composition comprising the mutant/s and/or variant/s. Insome embodiments, the mutant/s and/or variant/s used by the disclosedmethods may carry at least one mutation and display resistance and/orreduced sensitivity to cleavage and/or inactivation by at least one tPA,or any mutant or variant thereof. In some embodiments, the mutant maycarry at least one of the mutation that substitute the Arg³¹² residueand/or any amino acid residue adjacent to the Arg³¹² of the wild typeADAMTS-13 with a charged amino acid residue. The wild type ADAMTS-13 maycomprise the amino acid sequence as denoted by SEQ ID NO: 2, or anyvariants or derivatives thereof.

In certain embodiments, the mutant/s and/or variant/s suitable for themethod of the invention may be as the ADAMTS-13 mutant/s and/orvariant/s of the invention, specifically, any of the mutants describedabove, or any combinations and kit/s thereof. In some alternative oradditional embodiments, the methods disclosed herein may comprise thestep of administering to a subject treated with at least one serineprotease, a therapeutically effective amount of at least one ADAMTS-13mutant, any truncated variant thereof or any composition comprising themutants of the invention.

In certain embodiments, the serine protease may be tPA, uPA, plasmin,thrombin or granulocyte elastase or other plasminogen activators likestreptokinase or any functional fragments or variants thereof.

Still further, in some further embodiments, the method disclosed hereinmay comprise the step of administering to a subject treated with atleast anti-coagulation compound (e.g., heparin, Warfarin (Comadin),clexan and the like), a therapeutically effective amount of at least oneADAMTS-13 mutant, any truncated variant thereof or any compositioncomprising the mutants of the invention.

In yet some other embodiments, the methods of the present disclosure maybe applicable for any coagulation-related conditions as disclosed hereinbefore. In yet some further embodiments, the disclosed methods may beapplicable for any one of DVT, PE, AIS, AMI, TTP, DIC, HUS, and SLE.

Still further, as discussed herein, the presently disclosed method isbased on combined treatment regimen wherein the ADAMTS-13 mutant/sand/or variant/s discussed herein are administered to a patient that isalready treated with a fibrinolytic compound such as tPA. However, itshould be appreciated that the present disclosure further providesmethods where an affective amount of at least one fibrinolytic compound,for example tPA is administered to a subject treated with at least oneof the ADAMTS-13 mutant/s and/or variant/s of the present disclosure.

It should be understood that in some embodiments, all components areadministered simultaneously (e.g., at the same time) or consecutively(e.g., one right after the other). For example, at least one ADAMTS-13mutant and/or variant/s as disclosed herein may be administered priorto, after and/or simultaneously with administration of the at least onefibrinolytic agent or compound, specifically, tPA. Thus, according tosome embodiments, the methods of the invention may further encompassadministering to the treated subject at least one ADAMTS-13 mutantand/or variant/s as disclosed herein, for example, the ADAMTS-13 R312Kmutant and/or any variant thereof, prior to, after and/or simultaneouslywith administration of the at least one fibrinolytic agent or compound,specifically, tPA.

Still further, the present disclosure provides therapeutic methods andcompositions that administer an effective amount of the ADAMTS-13mutant/s and or variant/s disclosed herein, to a subject in needthereof. The terms “effective amount” or “sufficient amount” mean anamount necessary to achieve a selected result. The “effective treatmentamount” is determined by the severity of the disease in conjunction withthe preventive or therapeutic objectives, the route of administrationand the patient's general condition (age, sex, weight and otherconsiderations known to the attending physician). More specifically, theterm “effective amount” relates to the amount of an active agent presentin a composition, specifically, the ADAMTS-13 mutant/s and or variant/sof the invention as described herein that is needed to provide a desiredlevel of active agent in the bloodstream or at the site of action in anindividual to be treated to give an anticipated physiological responsewhen such composition is administered. The precise amount will dependupon numerous factors, e.g., the active agent, the activity of thecomposition, the delivery device employed, the physical characteristicsof the composition, intended patient use (i.e., the number of dosesadministered per day), patient considerations, and the like, and canreadily be determined by one skilled in the art, based upon theinformation provided herein. An “effective amount” of the ADAMTS-13mutant/s and or variant/s of the invention can be administered in oneadministration, or through multiple administrations of an amount thattotal an effective amount, preferably within a 24-hour period. It can bedetermined using standard clinical procedures for determiningappropriate amounts and timing of administration. It is understood thatthe “effective amount” can be the result of empirical and/orindividualized (case-by-case) determination on the part of the treatinghealth care professional and/or individual.

Still further, in some embodiments, an effective amount of the ADAMTS-13mutant/s and or variant/s may range between about 0.1 to about 0.9gr/day/kg. More specifically, between about 0.1 to about 0.2 gr perday/per kg, about 0.2 gr per day/per kg or between about 0.2 to about0.3 gr per day/per kg, about 0.3 gr per day/per kg or between about 0.3to about 0.4 gr per day/per kg, about 0.4 gr per day/per kg or betweenabout 0.4 to about 0.5 gr per day/per kg, about 0.5 gr per day/per kg orbetween about 0.5 to about 0.6 gr per day/per kg, about 0.6 gr perday/per kg or between about 0.6 to about 0.7 gr per day/per kg, about0.7 gr per day/per kg or between about 0.7 to about 0.8 gr per day/perkg, about 0.8 gr per day/per kg or between about 0.8 to about 0.9 gr perday/per kg, about 0.9 gr per day/per kg or between about 0.9 to aboutbut no more than 0.99 gr per day/per kg, and in some embodiments, lessthan 1 gr per day/per kg, for each of the ADAMTS-13 mutant/s and orvariant/s. In yet some further embodiments, an effective amount of theADAMTS-13 mutant/s and or variant/s may range between about 0.1 to about10 mg/day/kg. More specifically, between about 0.5 to about 8 mg perday/per kg, about 8 mg per day/per kg or between about 1 to about 7 mgper day/per kg, about 7 mg per day/per kg or between about 1.5 to about6 mg per day/per kg, about 6 mg per day/per kg or between about 2 toabout 5 mg per day/per kg, about 5 mg per day/per kg or between about2.5 to about 4.5 mg per day/per kg, about 4.5 mg per day/per kg orbetween about 3 to about 4 mg per day/per kg, about 4 mg per day/per kgor between about 0.1 to about 1 mg per day/per kg, about 1 mg perday/per kg or between about 0.5 to about 0.9 mg per day/per kg, about0.9 mg per day/per kg and in some embodiments, less than 10 mg perday/per kg, for each of the ADAMTS-13 mutant/s and or variant/s. In yetsome particular and non-limiting embodiments, an effective amount of theADAMTS-13 mutant/s and or variant/s is about 1 mg/kg/day. It should beappreciated however that the indicated effective doses per day, ordosage unit as discussed herein, may be given either in a singleadministration or in two or more administrations at several time-pointsover 24 hr. Still further, administration and doses are determined bygood medical practice of the attending physician and may depend onvarious general conditions of the subject in need.

The invention provides therapeutic methods and compositions for treatingspecific pathologic conditions and disorders. As used herein, “disease”,“disorder”, “condition” and the like, as they relate to a subject'shealth, are used interchangeably and have meanings ascribed to each andall of such terms. It should be appreciated that the invention providestherapeutic methods applicable for any of the disorders disclosed above,as well as to any condition or disease associated therewith. It isunderstood that the interchangeably used terms “associated”, “linked”and “related”, when referring to pathologies herein, mean diseases,disorders, conditions, or any pathologies which at least one of: sharecausalities, co-exist at a higher than coincidental frequency, or whereat least one disease, disorder condition or pathology causes the seconddisease, disorder, condition or pathology. More specifically, as usedherein, “disease”, “disorder”, “condition”, “pathology” and the like, asthey relate to a subject's health, are used interchangeably and havemeanings ascribed to each and all of such terms.

The terms “treat, treating, treatment” as used herein and in the claimsmean ameliorating one or more clinical indicia of disease activity byadministering a pharmaceutical composition of the invention in a patienthaving a pathologic disorder.

The term “treatment” as used herein refers to the administering of atherapeutic amount of the composition of the present invention which iseffective to ameliorate undesired symptoms associated with a disease, toprevent the manifestation of such symptoms before they occur, to slowdown the progression of the disease, slow down the deterioration ofsymptoms, to enhance the onset of remission period, slow down theirreversible damage caused in the progressive chronic stage of thedisease, to delay the onset of said progressive stage, to lessen theseverity or cure the disease, to improve survival rate or more rapidrecovery, or to prevent the disease form occurring or a combination oftwo or more of the above.

The term “prevention”, “prophylaxis” as used herein, includes theprevention or postponement of development of the disease, prevention orpostponement of development of symptoms and/or a reduction in theseverity of such symptoms that will or are expected to develop,preventing the occurrence or reoccurrence of the acute disease attacks.In yet some further embodiments, prophylaxis also encompasses anyreduction or attenuation of the susceptibility to develop the disease,and any reduction or inhibition of the occurrence or reoccurrence of thedisease. These further include ameliorating existing symptoms,preventing-additional symptoms and ameliorating or preventing theunderlying metabolic causes of symptoms.

The term “amelioration” as referred to herein, relates to a decrease inthe symptoms, and improvement in a subject's condition brought about bythe compositions and methods according to the invention, wherein saidimprovement may be manifested in the forms of inhibition of pathologicprocesses associated directly or indirectly with the coagulation, asdescribed herein, a significant reduction in their magnitude, or animprovement in a diseased subject physiological state.

The term “inhibit” and all variations of this term is intended toencompass the restriction or prohibition of the progress andexacerbation of pathologic symptoms or a pathologic process progress,said pathologic process symptoms or process are associated with.

The term “eliminate” relates to the substantial eradication or removalof the pathologic symptoms and possibly pathologic etiology, optionally,according to the methods of the invention described herein.

The terms “delay”, “delaying the onset”, “retard” and all variationsthereof are intended to encompass the slowing of the progress and/orexacerbation of a pathologic disorder or coagulation process and theirsymptoms slowing their progress, further exacerbation or development, soas to appear later than in the absence of the treatment according to theinvention.

More specifically, treatment or prevention include the prevention orpostponement of development of the disease, prevention or postponementof development of symptoms and/or a reduction in the severity of suchsymptoms that will or are expected to develop. These further includeameliorating existing symptoms, preventing-additional symptoms andameliorating or preventing the underlying metabolic causes of symptoms.It should be appreciated that the terms “inhibition”, “moderation”,“reduction” or “attenuation” as referred to herein, relate to theretardation, restraining or reduction of a process by any one of about1% to 99.9%, specifically, about 1% to about 5%, about 5% to 10%, about10% to 15%, about 15% to 20%, about 20% to 25%, about 25% to 30%, about30% to 35%, about 35% to 40%, about 40% to 45%, about 45% to 50%, about50% to 55%, about 55% to 60%, about 60% to 65%, about 65% to 70%, about75% to 80%, about 80% to 85% about 85% to 90%, about 90% to 95%, about95% to 99%, or about 99% to 99.9%.

With regards to the above, it is to be understood that, where provided,percentage values such as, for example, 10%, 50%, 120%, 500%, etc., areinterchangeable with “fold change” values, i.e., 0.1, 0.5, 1.2, 5, etc.,respectively.

The present invention relates to the treatment of subjects, or patients,in need thereof. By “patient” or “subject in need” it is meant anyorganism to whom the preventive and prophylactic combinations,composition/s, kit/s, and methods herein described is desired, includinghumans and domestic and/or wild mammals. More specifically, thetherapeutic methods disclosed herein are applicable for any subject. Insome particular embodiments, the methods of the invention may beparticularly applicable for a mammal (specifically, at least one of ahuman, Cattle, rodent, domestic pig (swine, hog), sheep, horse, goat,alpaca, lama and Camels), an avian, an insect, a fish, an amphibian, areptile, a crustacean, a crab, a lobster, a snail, a clam, an octopus, astarfish, a sea-urchin, jellyfish, and worms, specifically, a mammaliansubject. In some specific embodiments, the treated subject may be ahuman subject. The subject may be male or female, a child or an adult.In exemplary embodiments, the subject is an adult (e.g., at least 18years old). The present invention relates to the treatment of subjects,or patients, in need thereof. It should be further noted thatparticularly in case of human subject, administering of the compositionsof the invention to the patient includes both self-administration andadministration to the patient by another person. It should be understoodthat any suitable administration mode may be applicable for the presentdisclosure, specifically, any systemic or local administration. Stillfurther, any suitable route including intraperitoneal, subcutaneous,transcutaneous, topical, intramuscular, intraarticular, subconjunctival,or mucosal, e.g. oral, intranasal, or intraocular administration. Localadministration to the area in need of treatment may be achieved by, forexample, by local infusion during surgery, topical application, directinjection into the specific organ. More specifically, the ADAMTSTM-13mutant/s and/or variant/s, as well as compositions and combinedcompositions thereof used in any of the methods of the invention, may beadapted for administration by parenteral, intraperitoneal, transdermal,oral (including buccal or sublingual), rectal, topical (including buccalor sublingual), vaginal, intranasal and any other appropriate routes.Such formulations may be prepared by any method known in the art ofpharmacy, for example by bringing into association the active ingredientwith the carrier(s) or excipient(s).

As noted above, the present invention may involve the use of differentactive ingredients specifically the ADAMTS-13 mutant of the inventionand tPA, that may be administered through different routes, dosages andcombinations. More specifically, the treatment of coagulation associateddiseases with a combination of active ingredients may involve separateadministration of each active ingredient. Therefore, a kit providing aconvenient modular format for the combined therapy would allow thedesired or preferred flexibility in the above parameters.

Thus, in another aspect, the invention relates to a kit comprising:

First (a), at least one ADAMTS-13 mutant/s and/or variant (e.g.,truncated variant thereof), or any composition thereof, optionally, inat least one dosage form. In certain embodiment, the mutant may carry atleast one mutation, and displays resistance and/or reduced sensitivityto cleavage and/or inactivation by at least one tPA, or any mutant orvariant thereof.

The second component (b), may be at least one tPA or any functionalfragments or variants thereof, or any composition thereof, optionally,in at least one second dosage form. It should be understood however,that the present disclosure further encompasses kits that comprise inaddition to the ADAMTS-13 mutants disclosed herein, any other serinproteases as specified above, and/or any other anti-coagulatingcompounds (e.g., heparin, Warfarin (Comadin), clexan and the like).

In some embodiments, the mutant of the kits of the present disclosurecarries at least one mutation that may substitute the Arg³¹² residueand/or any amino acid residue adjacent to the Arg³¹² of the wild typeADAMTS-13 with a charged amino acid residue. The wild type ADAMTS-13 maycomprise the amino acid sequence as denoted by SEQ ID NO: 2.

In other embodiments, the mutant suitable for the kit of the inventionmay comprise substitution of residue 312, and/or any adjacent residue toany charged residue, specifically, any one of lysine, aspartic acid,glutamic acid or histidine. In some specific embodiments, the chargedamino acid may be lysine.

In some more specific embodiment, the mutant/s and/or variant/s of thekit of the invention may carry a mutation substituting the Arginine inposition 312 to lysine and may be designated R312K. In some embodiments,such mutant comprises the amino acid sequence as denoted by SEQ IDNO:11, or any derivatives or variants thereof. In yet some furtherembodiments, the mutant may be a truncated variant of the mutant of theinvention, that may comprise in some embodiments, the amino acidsequence as denoted by SEQ ID NO:5, or any derivatives or variantsthereof.

In yet some other embodiments, the mutant of the kit of the inventionmay be valine 313, and may carry a mutation substituting valine 313 withaspartic acid. In some embodiments, the mutant may be designated V313D.Still further, in some specific embodiments, such mutant may comprisethe amino acid sequence as denoted by SEQ ID NO: 14, or any derivativesor variants thereof. In yet some further embodiments, a truncatedvariant of such mutant may comprise the amino acid sequence as denotedby SEQ ID NO: 17.

In some specific embodiments, any mutant/s or variant/s of ADAMTS-13disclosed herein is suitable for the kits disclosed herein, with theproviso that the mutant and/or variant does not carry a mutationsubstituting the Arginine in position 312 to Alanine.

In some other embodiments, the kit of the invention may comprise: In afirst component (a), at least one ADAMTS-13 mutant, any truncatedvariant thereof, or any composition thereof. In certain embodiment, themutant may carry at least one mutation. More specifically, at least oneof the mutations in the mutant of the invention may be the substitutionof the Arg³¹² residue or any amino acid residue adjacent to the Arg³¹²of the wild type ADAMTS-13 with a charged amino acid residue. The wildtype ADAMTS-13 may comprise the amino acid sequence as denoted by SEQ IDNO: 2. The second component of the kit of the invention (b), maycomprise at least one serine protease or any functional fragments orvariants thereof, or any composition thereof. In some furtherembodiments, the serine protease may be any one of tPA, uPA, plasmin,thrombin or granulocyte elastase or other plasminogen activators likestreptokinase or any functional fragments or variants thereof.

According to some embodiments, the kit of the invention may furthercomprise container means for containing the different components of thekit of the invention or any dosage forms thereof. The term “container”as used herein refers to any receptacle capable of holding at least onecomponent of a pharmaceutical composition of the invention. Such acontainer may be any jar, vial or box known to a person skilled in theart and may be made of any material suitable for the componentscontained therein and additionally suitable for short- or long-termstorage under any kind of temperature. More specifically, the kitincludes container means for containing separate composition/s; such asa divided bottle or a divided foil packet however, the separatecompositions may also be contained within a single, undivided container.Typically, the kit includes directions for the administration of theseparate components, compounds or agents. As noted above, the kit formis particularly advantageous when the separate components, compounds oragents are preferably administered in different dosage forms (e.g., oraland parenteral), are administered at different dosage intervals, or whentitration of the individual components of the combination is desired bythe prescribing physician.

In yet some other embodiments, the kit of the invention may be for usein a method for the treatment, amelioration, inhibition or prophylaxisof any disease, disorder, or condition associated with coagulation in asubject in need thereof. It should be further appreciated that the kitsdisclosed herein may be applicable for any of the disclosed pathologies.

In some specific and non-limiting embodiments, the kits of the presentdisclosure may be applicable for any one of DVT, PE, AIS, AMI, TTP, DIC,HUS, SLE.

In a further aspect, the invention relates to at least one ADAMTS-13mutant, any truncated variant thereof or any composition comprising themutant, for use in a method of treatment, amelioration, inhibition orprophylaxis of a disease, disorder, or condition associated withcoagulation in a subject in need thereof. In some embodiments, themutant may carry at least one mutation and displays resistance and/orreduced sensitivity to cleavage and/or inactivation by at least one tPA,or any mutant or variant thereof.

In some embodiments, the mutant carries at least one mutation thatsubstitute the Arg³12 residue or any amino acid residue adjacent to theArg³12 of the wild type ADAMTS-13 with a charged amino acid residue. Itshould be noted that the wild type ADAMTS-13 comprises the amino acidsequence as denoted by SEQ ID NO: 2.

In some further embodiments, the charged amino acid residue of the atleast one ADAMTS-13 mutant for use according to the invention, may beany one of lysine, aspartic acid, glutamic acid or histidine.

In some specific embodiments, the charged amino acid residue of the atleast one ADAMTS-13 mutant for use according to the invention, may belysine.

In some more specific embodiments, the at least one ADAMTS-13 mutant foruse according to the invention, may carry a mutation substituting theArginine in position 312 to lysine and may be designated R312K. Stillfurther, in some embodiments, the mutant may comprise the amino acidsequence as denoted by SEQ ID NO:11 or any derivatives or variantsthereof. In yet some further embodiments, the truncated variant of themutant may comprise the amino acid sequence as denoted by SEQ ID NO:5 orany derivatives or variants thereof.

In yet another embodiment, the mutant for use according to the inventionmay be valine 313 and may carry a mutation substituting valine 313 withaspartic acid. In some embodiments, the mutant may be designated V313D.In some specific embodiments, such mutant may comprise the amino acidsequence as denoted by SEQ ID NO: 14, or any derivatives or variantsthereof. In yet some further embodiments, a truncated version of suchmutant may carry the amino acid sequence as denoted by SEQ ID NO: 17, orany derivatives or variants thereof.

In some further embodiments, the at least one ADAMTS-13 mutant for useaccording to the invention, may be as described above.

In some embodiments, the at least one ADAMTS-13 mutant for use accordingto the invention, may display resistance and/or decreased sensitivity tocleavage and/or inactivation by at least one tPA, or any mutant orvariant thereof.

In some embodiments, the at least one ADAMTS-13 mutant for use accordingto the invention, may display resistance to cleavage by at least oneserine protease or variant thereof. In certain embodiments, the serineprotease may be tPA, uPA, plasmin, thrombin or granulocyte elastase orother plasminogen activators like streptokinase or any functionalfragments or variants thereof.

In some further embodiments, the at least one ADAMTS-13 mutant for useaccording to the invention, refers to a mutant or any truncated variantthereof that display an increased activity.

In yet some further embodiments, the ADAMTS-13 mutant for use inaccordance with the present disclosure may display a prolonged half-liferelative to ADAMTS-13 wild type.

In yet some other embodiments, the at least one ADAMTS-13 mutant for useaccording to the invention, may be relevant to any disease, disorder, orcondition associated with coagulation. In some particular andnon-limiting embodiments, such disorders may be any one of DVT, PE, AIS,AMI, TTP, DIC, HUS, SLE.

Still further, the invention provides a therapeutically effective amountof at least one ADAMTS-13 mutant/s and/or variant/s (e.g., any truncatedvariant thereof) or any composition comprising the mutant for use in amethod for the treatment, amelioration, inhibition or prophylaxis of adisease, disorder, or condition associated with coagulation in a subjectin need thereof. It should be noted that the subject is a subject beingtreated with at least one tPA, or any mutant or variant thereof. In yetsome further specific embodiments, the mutant carries at least onemutation, and displays resistance and/or reduced sensitivity to cleavageand/or inactivation by at least one tPA, or any mutant or variantthereof. In some embodiments, the mutant carries at least one mutationthat in more specific embodiments, substitutes the Arg³¹² residue and/orany amino acid residue adjacent to said Arg³¹² of the wild typeADAMTS-13 with a charged amino acid residue.

In some embodiments, the ADAMTS-13 mutant provided for use in accordancewith the invention is any of the mutants disclosed by the invention.

In yet some further embodiments, the use according to the invention isspecifically applicable for subjects suffering from at least onedisease, disorder, or condition, specifically, any disease or disorderassociates with coagulation. In some embodiments, such disorders may beany one of DVT, PE, AIS, AMI, TTP, DIC, HUS, SLE.

Still further, in another aspect, the invention relates to a mutant ofADAMTS-13 that carries at least one mutation. In some embodiments, atleast one of the mutations in the mutants of the invention maysubstitute the Arginine in position 312 (Arg312) or in any amino acidresidue adjacent to the Arg312 of the wild type ADAMTS-13 with a chargedamino acid residue, or any truncated variant thereof. It should beunderstood that residue 312, as indicated herein refers to the aminoacid sequence of the wild type ADAMTS-13, that may comprise the aminoacid sequence as denoted by SEQ ID NO: 2, or any variants or derivativesthereof.

In yet some other aspect, the invention relates to a compositioncomprising an effective amount of at least one ADAMTS-13 mutant, or anytruncated variant thereof. In some embodiments, the mutant may carry amutation and at least one of the mutation/s may substitute the Arg³¹²residue or any amino acid residue adjacent to the Arg³¹² of the wildtype ADAMTS-13 with a charged amino acid residue. It should be notedthat the wild type ADAMTS-13 comprises the amino acid sequence asdenoted by SEQ ID NO: 2, and therefore the position 312, refers to SEQID NO: 2. In some embodiments, the composition may optionally furthercomprise at least one pharmaceutically acceptable carrier, diluent,excipient and/or additive.

In another aspect, the invention relates to a combined compositioncomprising a combination of at least one ADAMTS-13 mutant or anytruncated variant thereof, and at least one tPA or any functionalfragments or variants thereof. In some embodiments, the mutant may carryat least one mutation. The at least one of the mutation/s may substitutethe Arg³¹² residue or any amino acid residue adjacent to said Arg³¹² ofthe wild type ADAMTS-13 with a charged amino acid residue. It should benoted that the wild type ADAMTS-13 may comprise the amino acid sequenceas denoted by SEQ ID NO: 2, or any variants or derivatives thereof.Still further, in some embodiments, the composition may optionallyfurther comprise at least one pharmaceutically acceptable carrier,diluent, excipient and/or additive.

In a further aspect, the invention relates to a method for thetreatment, amelioration, inhibition or prophylaxis of a disease,disorder, or condition associated with coagulation in a subject in needthereof. In some embodiments, the method may comprise the step ofadministering to the subject a therapeutically effective amount of atleast one ADAMTS-13 mutant, any truncated variant thereof, or anycomposition or combined composition comprising the mutant of theinvention. In certain embodiments, the mutants of the invention maycarry at least one mutation. More specifically, at least one of themutation/s may substitute the Arg312 residue or any amino acid residueadjacent to the Arg312 of the wild type ADAMTS-13 with a charged aminoacid residue. It should be noted that the wild type ADAMTS-13 maycomprise the amino acid sequence as denoted by SEQ ID NO: 2.

In another aspect, the invention relates to a method for the treatment,amelioration, inhibition or prophylaxis of a disease, disorder, orcondition associated with coagulation in a subject in need thereof,comprising the step of administering to a subject treated with at leastone tissue plasminogen activator (tPA), or any mutant or variantthereof, a therapeutically effective amount of at least one ADAMTS-13mutant, any truncated variant thereof or any composition comprising themutant. In some embodiments, the mutant may carry a mutation, at leastone of the mutation/s may substitute the Arg³12 residue or any aminoacid residue adjacent to said Arg³¹² of the wild type ADAMTS-13 with acharged amino acid residue. The wild type ADAMTS-13 may comprise theamino acid sequence as denoted by SEQ ID NO: 2, or any variants orderivatives thereof.

In yet another aspect, the invention relates to a kit comprising: First(a), at least one ADAMTS-13 mutant, any truncated variant thereof, orany composition thereof. In certain embodiment, the mutant may carry atleast one mutation, at least one of the mutation/s may substitute theArg³¹² residue or any amino acid residue adjacent to the Arg³¹² of thewild type ADAMTS-13 with a charged amino acid residue. The said wildtype ADAMTS-13 may comprise the amino acid sequence as denoted by SEQ IDNO: 2. The second component (b), may be at least one tPA or anyfunctional fragments or variants thereof, or any composition thereof.

In a further aspect, the invention relates to at least one ADAMTS-13mutant, any truncated variant thereof or any composition comprising themutant, for use in a method of treatment, amelioration, inhibition orprophylaxis of a disease, disorder, or condition associated withcoagulation in a subject in need thereof. In some embodiments, themutant may carry at least one mutation. More specifically, at least oneof the mutation/s may substitute the Arg³¹² residue or any amino acidresidue adjacent to the Arg³12 of the wild type ADAMTS-13 with a chargedamino acid residue. It should be noted that the wild type ADAMTS-13comprises the amino acid sequence as denoted by SEQ ID NO: 2.

The invention further provides a therapeutically effective amount of atleast one ADAMTS-13 mutant, any truncated variant thereof or anycomposition comprising the mutant for use in a method for the treatment,amelioration, inhibition or prophylaxis of a disease, disorder, orcondition associated with coagulation in a subject in need thereof. Itshould be noted that the subject is a subject being treated with atleast one tPA, or any mutant or variant thereof. In yet some furtherspecific embodiments, the mutant carries at least one mutation, in morespecific embodiments, the mutation substitutes the Arg³12 residue or anyamino acid residue adjacent to said Arg³¹² of the wild type ADAMTS-13with a charged amino acid residue.

All definitions, as defined and used herein, should be understood tocontrol over dictionary definitions, definitions in documentsincorporated by reference, and/or ordinary meanings of the definedterms.

The term “about” as used herein indicates values that may deviate up to1%, more specifically 5%, more specifically 10%, more specifically 15%,and in some cases up to 20% higher or lower than the value referred to,the deviation range including integer values, and, if applicable,non-integer values as well, constituting a continuous range. In someembodiments, the term “about” refers to ±10%.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.” It must be notedthat, as used in this specification and the appended claims, thesingular forms “a”, “an” and “the” include plural referents unless thecontent clearly dictates otherwise.

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Multiple elements listed with“and/or” should be construed in the same fashion, i.e., “one or more” ofthe elements so conjoined. Other elements may optionally be presentother than the elements specifically identified by the “and/or” clause,whether related or unrelated to those elements specifically identified.Thus, as a non-limiting example, a reference to “A and/or B”, when usedin conjunction with open-ended language such as “comprising” can refer,in one embodiment, to A only (optionally including elements other thanB); in another embodiment, to B only (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of,” or, when usedin the claims, “consisting of,” will refer to the inclusion of exactlyone element of a number or list of elements. In general, the term “or”as used herein shall only be interpreted as indicating exclusivealternatives (i.e., “one or the other but not both”) when preceded byterms of exclusivity, such as “either,” “one of,” “only one of,” or“exactly one of” “Consisting essentially of,” when used in the claims,shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc. It shouldalso be understood that, unless clearly indicated to the contrary, inany methods claimed herein that include more than one step or act, theorder of the steps or acts of the method is not necessarily limited tothe order in which the steps or acts of the method are recited.

Throughout this specification and the Examples and claims which follow,all transitional phrases such as “comprising,” “including,” “carrying,”“having,” “containing,” “involving,” “holding,” “composed of,” and thelike are to be understood to be open-ended, i.e., to mean including butnot limited to. Specifically, it should understood to imply theinclusion of a stated integer or step or group of integers or steps butnot the exclusion of any other integer or step or group of integers orsteps. Only the transitional phrases “consisting of” and “consistingessentially of” shall be closed or semi-closed transitional phrases,respectively, as set forth in the United States Patent Office Manual ofPatent Examining Procedures. More specifically, the terms “comprises”,“comprising”, “includes”, “including”, “having” and their conjugatesmean “including but not limited to”. The term “consisting of” means“including and limited to”. The term “consisting essentially of” meansthat the composition, method or structure may include additionalingredients, steps and/or parts, but only if the additional ingredients,steps and/or parts do not materially alter the basic and novelcharacteristics of the claimed composition, method or structure.

It should be noted that various embodiments of this invention may bepresented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible sub ranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed sub ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 3, 4, 5, and 6. This appliesregardless of the breadth of the range. Whenever a numerical range isindicated herein, it is meant to include any cited numeral (fractionalor integral) within the indicated range. The phrases “ranging/rangesbetween” a first indicate number and a second indicate number and“ranging/ranges from” a first indicate number “to” a second indicatenumber are used herein interchangeably and are meant to include thefirst and second indicated numbers and all the fractional and integralnumerals there between.

As used herein the term “method” refers to manners, means, techniquesand procedures for accomplishing a given task including, but not limitedto, those manners, means, techniques and procedures either known to, orreadily developed from known manners, means, techniques and proceduresby practitioners of the chemical, pharmacological, biological,biochemical and medical arts.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable sub combination or as suitable in any other describedembodiment of the invention. Certain features described in the contextof various embodiments are not to be considered essential features ofthose embodiments, unless the embodiment is inoperative without thoseelements.

Various embodiments and aspects of the present invention as delineatedherein above and as claimed in the claims section below findexperimental support in the following examples.

Disclosed and described, it is to be understood that this invention isnot limited to the particular examples, methods steps, and compositionsdisclosed herein as such methods steps and compositions may varysomewhat. It is also to be understood that the terminology used hereinis used for the purpose of describing particular embodiments only andnot intended to be limiting since the scope of the present inventionwill be limited only by the appended claims and equivalents thereof.

The following examples are representative of techniques employed by theinventors in carrying out aspects of the present invention. It should beappreciated that while these techniques are exemplary of preferredembodiments for the practice of the invention, those of skill in theart, in light of the present disclosure, will recognize that numerousmodifications can be made without departing from the spirit and intendedscope of the invention.

EXAMPLES

Without further elaboration, it is believed that one skilled in the artcan, using the preceding description, utilize the present invention toits fullest extent. The following preferred specific embodiments are,therefore, to be construed as merely illustrative, and not limitative ofthe claimed invention in any way.

Experimental Procedures

Expression of MDTCS R312K in S2 Cells with pMT Based Plasmid and ProteinPurification

The plasmid had been transfected into S2 cells, and the cells wereselected with hygromycin B. The growing media was Schneider's DrosophilaMedium containing 10% of FBS, 0.5% of P/S (optional), 0.2 mg/ml ofhygromycin B. The expression media was Insect Xpress from. The S2 cellswere grown at room temperature, (best temperature is 26° C.). 1 vial(tube) of stock S2 cells is taken with expected plasmid transfected fromliquid N2. The cells were suspended and moved into a T25 flask. 5 ml (or4 ml) of growing media was added with repeated pipetting. The cells wereincubated at room temperature overnight. On the next day, change thefresh growing media is changed and the cells were incubated at roomtemperature for 3 day (±1 day) until the cell start to clump. The cellswere suspended by pipette, then moved into a 15-ml tube, spin down with1000 rpm for 5 min, and the supernatant was aspirated away. The cellswere re-suspended the cells 5 ml of fresh media, moved into a T75 flaskand media was added to final 15-20 ml. Incubate at room temperature. Thecell density was counted, based on the total volume. When the total cellnumber was 400×10⁶ or higher, the cells were spin down the cells. Thecells were re-suspended with 100 ml of Insect-Xpress media (Pen/Strepadded) and moved into a PETG flask. The cell density was checked daily.When the cell density was or higher than 20×10⁶ cells/ml, fresh Xpressmedia was added to final 500 ml (400-500 ml), The final cell density was4×10⁶ cells/ml or higher and the cells were shake. At same day, after2-3 hrs shake, CuSO4 was added to 0.5 mM and cells were shake. Celldensity was checked, when it was higher than 30×10⁶ cells/ml (3-5 days),the cells were spin down with 2000 rpm for 10 min, the supernatant wascollected. A filter with 0.2 μm filter Unit was used.

At the C-terminus, the DMTCS R311K construct has a triple-FLAG tag. Forpurification, the supernatant was loaded on an anti-FLAG column(commercially available). The column was balanced with either InsectXpress media or PBS before loading the sample. The loading speed wasdetermined by natural flow, usually loading no more than 100-200 ml foran hour to a few hours at cold room. Wash column with PBS, run 10×column volume. The sample was eluted either with 0.1M Glycine-HCl pH2.6-2.8 and neutralized with ⅛ volume of Tris-HCl pH 8.0, or inaccordance with the column supplier recommendations. The neutralizedMDTCS R311K protein was collected and concentrated with the simultaneousbuffer change for PBS on Amicon Ultra Centrifugal Filters 50K. Theprotein concentration was checked at OD₂₈₀ 1.186=1 mg/ml.

Construction of the Truncated R312K Mutant:

The mutated R312K truncated sequence starts from amino acid residue 75in the mature ADAMTS-13 sequence (first 74 amino acids cut) and ends atthe amino acid residue 685 of the mature ADAMTS-13 sequence (see aminoacid sequence as denoted by SEQ ID NO: 5). The WT truncated ADAMTScomprise an amino acid sequence as denoted by SEQ ID NO: 7, encoded bythe nucleic acid sequence as denoted by SEQ ID NO: 6. In order toconstruct the truncated R312K mutant the following primers were used:the forward primer as denoted by SEQ ID NO: 8 and the reverse primer asdenoted by SEQ ID NO: 9, as well as the AntiK primer as denoted by SEQID NO: 10. Primer SEQ ID NO: 20 was used to mutate R312 to A.

Example 1

tPA Cleaves and Inactivated ADAMTS-13

To examine the capacity of tPA to cleave ADAMTS-13, fetal liver cellslysates (A549 cells) and human plasma which express the native ADAMTS-13were incubated in the absence or presence of tPA (100 nM) for 2 hrs at37° C. To exclude the possibility that tPA cleaves ADAMTS-13 viaplasminogen activation to plasmin, the lysates and plasma were incubatedin tPA with the serine protease—aprotinin—a plasmin inhibitor (1 μM).Western blots were performed using monoclonal anti-ADAMTS-13 antibodyEPR6132 from Abcam. FIG. 1 A-B show that tPA cleaves ADAMTS-13 in celllysates (A) and human plasma (B), and the cleavage is unaffected by theplasmin inhibitor (tPA+Ap), indicating that ADMATS-13 is not cleaved byplasmin, and most likely, cleaved by tPA. To investigate thephysiological relevance of these observations, it was asked whether tPAregulates ADAMTS-13 activity in vitro and in vivo and whether the tPAADAMTS-13 interactions affects vWF activity in vivo. The ADAMTS-13activity was measured in cell lysates and human plasma, in the presenceand absence of tPA (100 nM). Indeed, it was shown that ADAMTS-13activity is significantly decreased in the presence of tPA, and thedecrease in the ADAMTS-13 activity was unaffected by aprotonin (FIG.1C). The ADAMTS-13 and vWF activities were further measured in WT andtPA−/− mice. Plasma ADAMTS-13 activity measured by ELISA wassignificantly higher (p<0.05) and vWF activity was lower (p<0.05) intPA−/− mice (FIG. 2 ).

The data presented in FIGS. 1 and 2 support the contention that, underphysiological conditions, tPA cleaves and inactivate ADAMTS-13 andsubsequently increases vWF activity.

Example 2

Regulation of ADAMTS-13 Activity

Human ADAMTS-13 is a multidomain protein (FIG. 3A) with Metalloproteasedomain (MP), Disinterring-like domain (Disin), 8 Thrombospondin type-1domains (1-8), Cysteine-rich domain (Cys), Spacer domain (S) and 2 CUBdomains. The metalloprotease domain contains the catalytic site ofADAMTS-13 that cleaves vWF. Since it appears that tPA inhibits thecatalytic activity of ADAMTS-13, it suggests that it affects its Nterminal domains, that contains the metalloprotease domain where thecatalytic activity is performed.

A truncated form of trADAMTS-13 containing the MP (M), Disin (D), 8Thrombospondin (T), Cys (C), (S) domains, indicated herein as MDTCSsubunits was therefore synthetized, creating a recombinant truncatedADAMTS-13 variant (trADAMTS-13), that contains MP domain (FIG. 3B).

trADAMTS-13 has the expected MW, of about 75 KDa (FIG. 4A), is fullyactive as the ADAMTS-13 WT (FIG. 4B), and in the presence of tPA, it iscleaved (FIG. 4C). As expected, the activity of both trADAMTS-13 andADAMTS-13 WT was reduced in the presence of tPA (FIG. 4B). Similarly,the inactivation of trADAMTS-13 by tPA prevented the proteolysis ofULvWV multimers present in human plasma (FIG. 4D), indicating thattrADAMTS-13 cleaves vWF and that tPA prevents such an effect. Theseresults clearly demonstrate that the truncated ADAMTS-13 variant retainsits ability to proteolytically inactivate ULvWF, and moreover, itretains the sensitivity to cleavage and/or inactivation by tPA.

Example 3

Delineation of the ADAMTS-13 Cleavage Site

Mass spectroscopy was used to identify the amide bond in ADAMTS-13cleaved by tPA. trADAMTS-13 was incubated with tPA, the degradationproducts were separated on SDS-PAGE (FIG. 5 ), individual bands wereexcised and their amino acid sequences were determined. Analysis of thesequence showed that tPA cleaves ADAMTS-13 at R³¹²-V³¹³, analogous toits cleavage site in plasminogen (Blood Coagul Fibrinolysis. 1992 3(5):605-614).

Example 4

Creation of variants that perturbs the R³¹²V³¹³ bond in ADAMTS-13

Based on the results presented above, the inventors hypothesized that asingle mutation that perturbs the R³¹²-V³¹³ bond in ADAMTS-13 wouldproduce a variant that would be resistant to cleavage by tPA. Suchmutant is expected to retain its ability to cleave vWF and by that, toimprove the trADAMTS-13 in-vivo activity.

To test this hypothesis, truncated recombinant ADAMTS-13 variants(trADAMTS-13) were next synthetized, with a single mutation where R312was replaced by: (1) Lysine (K) (trADAMTS-13^(R312K)), (2) Alanine (A)(trADAMTS-13^(R312A)) or (3) by replacing V³¹³ with Alanine A(trADAMTS-13^(V313A)) or (4) with Aspartate (D) (trADAMTS-13^(V313D))All the variants were cloned and expressed in S2 cells.

All the variants were analyzed by SDS-PAGE (FIG. 6A) and their catalyticactivity on vWF was compared (FIG. 6B).

As shown by FIG. 6B, although all of the four variants migrate as asingle chain molecule at the same Molecular Weight of the un-mutatedvariant (FIG. 6A), they exhibited different levels of catalytic activity(FIG. 6B).

trADAMTS-13^(R312K) was the most potent variant exhibiting enhancedproteolytic activity on vWF (FIG. 6B), in comparison with the un-mutatedprotein trADAMTS-13 WT. In line with the data presented by Europeanpatent application EP 2172544 [18], trADAMTS-13^(R312A) catalyticactivity was not affected significantly by replacing R³¹² with A (FIG.6B). Replacing V³¹³ with A (trADAMTS-13^(V313A)), also had nosignificant effect on its proteolytic activity on vWF (FIG. 6B). Incontrast, replacing V³¹³ with D (trADAMTS-13^(V313D)) decreased by morethan 50% the proteolytic activity of the resultant ADAMTS-13 variant onvWF (FIG. 6B).

However, surprisingly, substitution to lysine in position 312, led toloss of sensitivity to tPA. Specifically, trADAMTS-13^(R312K) wasresistant to cleavage by tPA under conditions where trADAMTS-13 wasproteolyzed completely (FIG. 7A) and lost its catalytic activity (FIG.7B).

The half-life of the truncated forms of ADAMTS-13 WT and mutant weremeasured. More specifically, trADAMTS-13 WT or trADAMTS-13^(R312K) (1 mgin saline) were injected IP to WT mice (n=245 in each group). Each groupof mice was divided into five sub-groups (n=5 in each group). Animalswere scarified at 5, 30, 60, 180 and 360 minutes after injection, andADAMTS-13 activity was determined. A group of five mice injected withsaline was used as a control determining time 0. The inventors foundthat the t½ of the trADAMTS-13 was 132.8±41.3 min and that oftrADAMTS-13^(R312K) was 344.6±131.1 min. Thus, the half-life oftruncated ADAMTS-13 mutant is significantly longer than the truncated WTor the full-length WT molecules. These data indicate that the truncatedvariant display an extended activity time and is therefore suitable forevents like DVT as well as from ischemic stroke.

Examining the effect of tPA on the catalytic activity of the 4trADAMTS-13 variants, revealed that trADAMTS-13^(R312K) andtrADAMTS-13^(V313D) variants were resistant to inactivation by tPA. Incontrast, tPA inhibited the proteolytic activity of trADAMTS-13^(R312A)and trADAMTS-13^(V313A) variants on vWF (FIG. 8 ).

In summary, mutations in the R³¹²-V³¹³ site clearly affect the catalyticactivity of ADAMTS-13. Although all of the tested variants maintainedcatalytic activity on vWF, trADAMTS-13^(R312K) variant is the mostpotent and is totally resistant to inactivation by tPA.

Example 5

Effect of Autoantibodies from TTP Plasma on trADAMTS-13^(R312K) Activity

Anti-ADAMTS-13 autoantibodies cause severe enzyme deficiency. ADAMTS-13deficiency causes the loss of regulation of vWF multimeric size andplatelet function, which results in the formation of disseminatedmicrovascular platelet microthrombi. Anti-ADAMTS-13 autoantibodiesrecognize mainly the N-terminal domains of the enzyme (Thomas et al.EBioMedicine 2015 2, 942-952), domains that are included in trADAMTS-13.R312 was shown to be one of the targets of the anti ADAMTS-13 antibodies(European patent application EP2172544 [18]). Furthermore, thispublication indicates that only replacement with uncharged amino acidcould reduce the antigenicity of ADAMTS-13 [18]. To examine the effectof anti ADAMTS-13 autoantibodies on the catalytic activity of the abovedescribed trADAMTS-13 variants, trADAMTS-13 WT and all of the fourvariants of the invention were incubated with plasma from patients withacquired ADAMTS-13 deficiency and <10% ADAMTS-13 activity for 2 hours.As expected, TTP plasma totally neutralized trADAMTS-13 WT.Surprisingly, trADAMTS-13^(R312K) was resistant to anti ADAMTS-13autoantibodies (FIG. 9A-9B). The substantial resistance to inhibition oftrADAMTS-13^(R312K) was shown around 2 hours up to 24 hours followingincubation with TTP plasma (FIG. 9B). In addition, trADAMTS-13^(R312A)also appeared to be resistant to anti ADAMTS-13 autoantibodies. Incontrast, trADAMTS-13V³13^(A) and trADAMTS-13V³13^(D) maintained theirsusceptibility to inhibition by the anti ADAMTS-13 autoantibodies (FIG.9A).

Example 6

Thrombolytic Effect of trADAMTS-13 Variants on VWF-Rich Thrombus

To test the therapeutic potential of ADAMTS-13 variants, a mouse modelthat recapitulates acute ischemic via thrombotic occlusion of thecarotid artery after topical application of FeCl3, was next used.Histological analysis of the occlusive thrombi formed by topicalapplication of FeCl3 demonstrated that they are rich in VWF (Denorme etal. Blood 2016 127:2337-2345).

As previously reported (Denorme et al. Blood 2016 127:2337-2345),thrombus formation was initiated by topical application of FeCl3 using alarger filter paper (0.53-1.5 mm) saturated with 20% FeCl3.

Sixteen weeks old C57B/6 mice were subjected to carotid arteryocclusion. The left common carotid artery was isolated, and a vascularflow probe (Transonic Systems, NY) was applied to monitor blood flow.FeCl3 was applied as reported (Denorme et al. Blood 2016 127:2337-2345).The time required to form an occlusive thrombus, is defined as the timerequired to drop blood flow below 25%, after FeCl3 application. Fiveminutes after occlusion, mice were given intravenous injection of tPA(0.5 mg/kg) alone or together with increasing doses of ADAMTS-13variants, trADAMTS-13 WT or trADAMTS-13^(R312K) (0.5, 1 or 5 mg/kgeach), and blood flow restoration was monitored for 120 minus. In theuntreated group (control), no improvement in blood flow was detected inblood flow even after 120 minutes of monitoring. FIG. 10 shows that timeto reperfusion, was improved significantly by the co-administration oftPA and trADAMTS-13. FIG. 10 also shows that trADAMTS-13^(R312K) wasmore effective than the un-mutated variant. Moreover, this figure showssynergistic activity when combining tPA with the ADAMTS-13 mutant of theinvention.

Example 7

Delineation of the Anti-Thrombotic Activity of trADAMTS-13 In Vivo

Since it appears that the anti-vWF activity of ADAMTS-13 is limited bytPA, it was hypothesized that by overcoming such limitation ADAMTS-13thrombolytic/anti-coagulation activities would be improved. To test thishypothesis in in vivo setting, three sets of experiments were performed.First, the effect of tPA on ADAMTS-13 activity was examined followed byvWF concentration and activity. WT and tPA−/− mice were injected IP withtrADAMTS-13 WT or trADAMTS-13^(R312K). Two hours later, plasma ADAMTS-13activity and concentration of vWF were assessed. Mice injected withtrADAMTS-13^(R312K) retained greater capacity to proteolyze vWF thanthose given trADAMTS-13 WT (FIGS. 11A and 11C) and respectively, theconcentration of vWF in trADAMTS-13^(R312K) was lower as well (FIG.11B). The activity of the variant trADAMTS-13^(R312K) and in correlationthe vWF weight were substantial similar in both tPA−/− mice and WT mice,whereas the activity of trADAMTS-13 WT was higher in the tPA−/− micecompared to WT mice (FIGS. 11A and 11B). Second, the effect of tPAresistance of ADAMTS-13 on the formation of venous clots was examinedusing an inferior vena cava (IVC) stasis model. WT mice were given an IPinjection of trADAMTS-13 WT or trADAMTS-13^(R312K) one hour beforeinducing clot formation. trADAMTS-13^(R312K) reduced clot size to asignificantly greater extent than trADAMTS-13 (FIG. 12B, p<0.05). Third,the effect on bleeding two hours after IP of trADAMTS-13 WT ortPA-resistant trADAMTS-13^(R312K) was examined in the tail cut modelbleeding (Abu-Fanne et al. Blood. 2019 Jan. 31; 133(5):481-493).Although both truncated, ADAMTS-13 variants increased the bleeding time,trADAMTS-13^(R312K) caused a greater prolongation in the bleeding timethan trADAMTS-13 WT (FIG. 13 ).

Example 8

Soluble Fibrin Reverses tPA-Mediated Inactivation of trADAMTS-13

tPA is a well-established fibrinolytic protein. However, the surprisingresults showing that tPA-mediate ADAMTS-13 inactivation, raised thequestion whether there may be a potential procoagulant function of tPA.

It is also well-established the that the plasminogen activator activityof tPA is almost undetectable in the absence of fibrin (Hoylaerts M,Rijken D C, Linjen H R, Collen D 1982, Journal of Biological Chemistry,257, 2912-2919). Therefore, to examine how these two seemingly opposingactivities are regulated, the effect of soluble fibrin was examined ontPA-mediated inactivation of trADAMTS-13. The activity of trADAMTS-13 WTwas measured following incubation for 4 hr in soluble fibrin or PBS,without or with tPA. The addition of soluble fibrin reversed thetPA-mediated reduction in ADAMTS-13 activity (FIG. 14A). Similarly, tomeasure the activity of wild type full length ADAMTS-13 in the presenceof fibrin and tPA, serum from WT mice was incubated with tPA without orwith fibrin. Similarly, the addition of fibrin, reversed thetPA-mediated inactivation in ADAMTS-13 activity (FIG. 14B).

These results show that fibrin down-regulates the potentiallyprocoagulant role and upregulates the fibrinolytic actions of tPA.

1-59. (canceled)
 60. A mutant and/or variant of a disintegrin andmetalloproteinase with a thrombospondin type 1 motif, member 13(ADAMTS-13) that carries at least one mutation, or any truncated variantthereof, wherein said mutant displays resistance and/or reducedsensitivity to cleavage and/or inactivation by at least one tissueplasminogen activator (tPA), or any mutant or variant thereof.
 61. TheADAMTS-13 mutant and/or variant according to claim 60, wherein saidmutant, variant or any truncated variant carries at least one mutationthat substitutes the Arginine in position 312 (Arg312) of the wild typeADAMTS-13 and/or any amino acid residue adjacent to said Arg312, with acharged amino acid residue, optionally, wherein said charged amino acidresidue is any one of lysine, aspartic acid, glutamic acid or histidine.62. The ADAMTS-13 mutant and/or variant, according to claim 60, or anytruncated variant thereof, wherein said mutant carries at least onemutation that substitutes the Arginine in position 312 to lysine, isdesignated R312K, and wherein at least one of: (a) said mutant comprisesthe amino acid sequence as denoted by SEQ ID NO:11, or any derivativesor variants thereof; and (b) said truncated variant of said mutantcomprises the amino acid sequence as denoted by SEQ ID NO:5, or anyderivatives or variants thereof, and optionally, wherein said adjacentamino acid residue is valine 313, optionally, said mutant carries amutation that substitutes valine 313 with aspartic acid.
 63. TheADAMTS-13 mutant and/or variant according to claim 60, wherein saidmutant or any truncated variant thereof display an increased activity,optionally, said mutant or any truncated variant thereof display aprolonged half-life relative to ADAMTS-13 wild type.
 64. A compositioncomprising an effective amount of the at least one ADAMTS-13 mutantaccording to claim 60, and/or variant that carries at least onemutation, said composition optionally further comprises at least onepharmaceutically acceptable carrier, diluent, excipient and/or additive.65. A combined composition comprising a combination of at least oneADAMTS-13 mutant according to claim 60, and/or variant and at least onetPA or any functional fragments or variants thereof, wherein saidmutant, variant or any truncated variant thereof, carries at least onemutation and displays resistance and/or reduced sensitivity to cleavageand/or inactivation by at least one tPA, or any mutant or variantthereof.
 66. The combined composition according to claim 65, whereinsaid composition further comprises fibrin.
 67. A method for thetreatment, amelioration, inhibition or prophylaxis of a disease,disorder, or condition associated with coagulation in a subject in needthereof, the method comprising the step of administering to said subjecta therapeutically effective amount of at least one ADAMTS-13 mutantand/or variant, or any truncated variant thereof, or any composition orcombined composition comprising said mutant, wherein said mutant carriesat least one mutation and displays resistance and/or reduced sensitivityto cleavage and/or inactivation, by at least one tPA, or any mutant orvariant thereof.
 68. The method according to claim 67, wherein saidmutant and/or variant or any truncated variant thereof carries at leastone mutation that substitutes the Arg312 residue of the wild typeADAMTS-13, and/or any amino acid residue adjacent to said Arg312, with acharged amino acid residue, optionally, said charged amino acid residueis any one of lysine, aspartic acid, glutamic acid or histidine.
 69. Themethod according to claim 67, wherein said mutant, variant or anytruncated variant thereof carries a mutation substituting the Argininein position 312 to lysine and is designated R312K, wherein at least oneof: (a) said mutant comprises the amino acid sequence as denoted by SEQID NO:11, or any derivatives or variants thereof, and (b) said truncatedvariant of said mutant comprises the amino acid sequence as denoted bySEQ ID NO:5, or any derivatives or variants thereof, optionally, saidadjacent amino acid reside is valine 313, optionally, said mutant,variant, or any truncated variant thereof carries a mutation thatsubstitutes valine 313 with aspartic acid.
 70. The method according toclaim 67, wherein at least one of: (a) said mutant, variant or anytruncated variant thereof or any truncated variant thereof display anincreased activity; (b) said mutant, variant or any truncated variantthereof or any truncated variant thereof displays a prolonged half-liferelative to ADAMTS-13 wild type; and (c) wherein said disease, disorder,or condition is at least one of deep venous thrombosis (DVT), pulmonaryemboli (PE), acute ischemic stroke (AIS), acute myocardial function(AMI), thrombotic thrombocytopenic purpura (TTP), disseminatedintravascular coagulation (DIC), hemolytic-uremic syndrome (HUS),cerebral infarction or systemic lupus erythematosus (SLE).
 71. Themethod according to claim 67, wherein: (a) said method further comprisesthe step of administering to said subject, a therapeutically effectiveamount of at least one tPA or any functional fragments or variantsthereof or any composition thereof; or (b) said method comprising thestep of administering to said subject, a therapeutically effectiveamount of a combined composition comprising a combination of at leastone ADAMTS-13 mutant and/or any truncated variant thereof, and at leastone tPA or any functional fragments or variants thereof.
 72. The methodaccording to claim 67, wherein said subject is a subject treated with atleast one tPA, or any mutant, variant or any truncated variant thereof,a therapeutically effective amount of at least one ADAMTS-13 mutant, anytruncated variant thereof or any composition comprising said mutant,wherein said mutant carries at least one mutation and displaysresistance and/or reduced sensitivity to cleavage and/or inactivation byat least one tPA, or any mutant or variant thereof, optionally, whereinsaid disease, disorder, or condition is any one of DVT, PE, AIS, AMI,TTP, DIC, HUS, SLE.
 73. A kit comprising: (a) at least one ADAMTS-13mutant and/or variant and/or any truncated variant thereof, or anycomposition thereof, optionally, in a first dosage form, wherein saidmutant or any truncated variant thereof, carries at least one mutationand displays resistance and/or reduced sensitivity to cleavage by atleast one tPA, or any mutant or variant thereof; and (b) at least onetPA or any functional fragments or variants thereof, or any compositionthereof, optionally, in a second dosage form.
 74. The kit according toclaim 73, wherein said mutant and/or variant or any truncated variantthereof, carries at least one mutation that substitutes the Arg312residue and/or any amino acid residue adjacent to said Arg312 of thewild type ADAMTS-13 with a charged amino acid residue, optionally,wherein said charged amino acid residue is any one of lysine, asparticacid, glutamic acid or histidine.
 75. The kit according to claim 73,wherein said mutant, variant, or any truncated variant thereof, carriesa mutation substituting the Arginine in position 312 to lysine and isdesignated R312K, wherein at least one of: (a) said mutant comprises theamino acid sequence as denoted by SEQ ID NO:11, or any derivatives orvariants thereof, and (b) said truncated variant of said mutantcomprises the amino acid sequence as denoted by SEQ ID NO:5, or anyderivatives or variants thereof, optionally, wherein said adjacent aminoacid reside is valine 313, and optionally, said mutant carries amutation substituting valine 313 with aspartic acid.
 76. The kitaccording to claim 73, adapted for use in a method for the treatment,amelioration, inhibition or prophylaxis of a disease, disorder, orcondition associated with coagulation in a subject in need thereof,optionally, said disease, disorder, or condition is any one of DVT, PE,AIS, AMI, TTP, DIC, HUS, SLE.
 77. A mutant of a disintegrin andmetalloproteinase with a thrombospondin type 1 motif, member 13(ADAMTS-13) that carries at least one mutation according to claim 60,wherein said at least one mutation substitutes the Arginine in position312 (Arg312) of the wild type ADAMTS-13 and/or any amino acid residueadjacent to said Arg312, with a charged amino acid residue, or anytruncated variant thereof, or a composition comprising an effectiveamount of said mutant, said composition optionally further comprises atleast one pharmaceutically acceptable carrier, diluent, excipient and/oradditive.
 78. A combined composition or kit comprising: (a) at least oneADAMTS-13 mutant according to claim 77, or any truncated variantthereof, (b) and at least one tPA or any functional fragments orvariants thereof, said composition or kit optionally further comprisesat least one pharmaceutically acceptable carrier, diluent, excipientand/or additive.
 79. A method for the treatment, amelioration,inhibition or prophylaxis of a disease, disorder, or conditionassociated with coagulation in a subject in need thereof, the methodcomprising the step of administering to said subject a therapeuticallyeffective amount of at least one ADAMTS-13 mutant according to claim 77,any truncated variant thereof, or any composition or combinedcomposition comprising said mutant optionally, wherein said subject is asubject treated with at least one tPA, or any mutant or variant thereof,a therapeutically effective amount of at least one ADAMTS-13 mutant, anytruncated variant thereof or any composition comprising said mutant.